Therapeutic Nuclease Compositions and Methods

ABSTRACT

Hybrid nuclease molecules and methods for treating an immune-related disease or disorder in a mammal, and a pharmaceutical composition for treating an immune-related disease in a mammal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/822,215, filed Mar. 11, 2013, which claims priority to U.S.Provisional Application No. 61/480,961, filed Apr. 29, 2011 and U.S.Provisional Application No. 61/617,241, filed Mar. 29, 2012, andNational Stage of International Application No. PCT/US2012/035614, filedApr. 27, 2012.

This application is also related to: International Patent ApplicationNo. PCT/US2010/055131, filed Nov. 2, 2010; U.S. Provisional ApplicationNo. 61/257,458, filed Nov. 2, 2009; and U.S. Provisional Application No.61/370,752, filed Aug. 4, 2010.

The entire disclosures of the foregoing applications are herebyincorporated by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants NS065933and AR048796 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Dec. 20, 2018, is named42456_US_Sequence_Listing.txt, and is 182 kilobytes in size.

BACKGROUND

Excessive release of (ribo)nucleoprotein particles from dead and dyingcells can cause lupus pathology by two mechanisms: (i) Deposition or insitu formation of chromatin/anti-chromatin complexes causes nephritisand leads to loss of renal function; and (ii) nucleoproteins activateinnate immunity through toll-like receptor (TLR) 7, 8, and 9 as well asTLR-independent pathway(s). Release of nucleoproteins can serve as apotent antigen for autoantibodies in SLE, providing amplification of Bcell and DC activation through co-engagement of antigen receptors andTLRs. Thus, there exists a need for a means to remove inciting antigensand/or attenuate immune stimulation, immune amplification, and immunecomplex mediated disease in subjects in need thereof.

SUMMARY

Disclosed herein is a hybrid nuclease molecule comprising a firstnuclease domain and a modified Fc domain, wherein the first nucleasedomain is operatively coupled to the Fc domain. The Fc domain ismodified such that the molecule has reduced cytotoxicity relative to ahybrid nuclease molecule having an unmodified Fc domain. In someembodiments, the hybrid nuclease molecule has an Fc domain which hasbeen modified to decrease binding to Fcγ receptors, complement proteins,or both. In some emboidments, the hybrid nuclease molecule has reducedcytotoxity at least 1, 2, 3, 4, or 5-fold compared to a control moleculee.g., a hybrid nuclease molecule without a modified Fc domain.

In some embodiments, the hybrid nuclease molecule further includes afirst linker domain, and the first nuclease domain is operativelycoupled to the modified Fc domain by the first linker domain.

In some aspects, a hybrid nuclease molecule includes a modified Fcdomain which is a mutant, IgG1 Fc domain. In some aspects, a mutant Fcdomain comprises one or more mutations in the hinge, CH2, and/or CH3domains. In some aspects, the Fc domain comprises an amino acid sequencehaving one or more of the mutations P238S, P331S, SCC, SSS (residues220, 226, and 229), G236R, L328R, L234A, and L235A. In some aspects, amutant Fc domain includes a P238S mutation. In some aspects, a mutant Fcdomain includes a P331S mutation. In some aspects, a mutant Fc domainincludes a P238S mutation and a P331S mutation. In some aspects, amutant Fc domain comprises P238S and/or P331S, and may include mutationsin one or more of the three hinge cysteines. In some aspects, a mutantFc domain comprises P238S and/or P331S, and/or one or more mutations inthe three hinge cysteines. In some aspects, a mutant Fc domain comprisesP238S and/or P331S, and/or mutations in one of the three hinge cysteines(located at residue 220 by EU numbering) to SCC (wherein CCC refers tothe three cysteines present in the wild type hinge domain). In someaspects, a mutant Fc domain comprises P238S and/or P331S, and/ormutations in the three hinge cysteines (located at residues 220, 226 and229 by EU numbering) to SSS. In some aspects, a mutant Fc domaincomprises P238S and P331S and mutations in the three hinge cysteines. Insome aspects, a mutant Fc domain comprises P238S and P331S and SCC. Insome aspects, a mutant Fc domain comprises P238S and P331S and SSS. Insome aspects, a mutant Fc domain includes P238S and SCC. In someaspects, a mutant Fc domain includes P238S and SSS. In some aspects, amutant Fc domain includes P331S and SCC. In some aspects, a mutant Fcdomain includes P331S and SSS. In some aspects, a mutant Fc domainincludes mutations in one or more of the three hinge cysteines. In someaspects, a mutant Fc domain includes mutations in the three hingecysteines. In some aspects, a mutant Fc domain includes a mutation inthe three hinge cysteines to SCC. In some aspects, a mutant Fc domainincludes mutations in the three hinge cysteines to SSS. In some aspects,a mutant Fc domain includes SCC. In some aspects, a mutant Fc domainincludes SSS.

In some aspects, a nucleic acid encoding a mutant Fc domain is as shownin SEQ ID NO:59. In some aspects, a mutant Fc domain is as shown in SEQID NO:60. In some aspects, a nucleic acid encoding a mutant Fc domain isas shown in SEQ ID NO:71. In some aspects, a mutant Fc domain is asshown in SEQ ID NO:72. In some aspects, a nucleic acid encoding a mutantFc domain is as shown in SEQ ID NO:73. In some aspects, a mutant Fcdomain is as shown in SEQ ID NO:74. In some aspects, a nucleic acidencoding a mutant Fc domain is shown in SEQ ID NO:75. In some aspects, amutant Fc domain is as shown in SEQ ID NO:76. In some aspects, a nucleicacid encoding a mutant Fc domain is as shown in SEQ ID NO:87. In someaspects, a mutant Fc domain is as shown in SEQ ID NO:88. In someaspects, a nucleic acid encoding a mutant Fc domain is as shown in SEQID NO:89. In some aspects, a mutant Fc domain is as shown in SEQ IDNO:90.

In some aspects, a hybrid nuclease molecule comprises a wild-type, humanRNase1 domain linked to a mutant, human IgG1 Fc domain comprising SCC,P238S, and P331S, or to a mutant, human IgG1 Fc domain comprising SSS,P238S, and P331S. In some aspects, a nucleic acid encoding a hybridnuclease molecule is as shown in SEQ ID NO: 61, 77, or 91. In someaspects, a hybrid nuclease molecule is as shown in SEQ ID NO:209, 62,78, 92, or 94.

In some aspects, a hybrid nuclease molecule comprises a wild-type, humanRNase1 domain linked via a (Gly₄Ser)₄ linker domain to a mutant, humanIgG1 Fc domain comprising SCC, P238S, and P331S, or to a mutant, humanIgG1 Fc domain comprising SSS, P238S, and P331S. In some aspects, anucleic acid encoding a hybrid nuclease molecule is as shown in SEQ IDNO: 63, or 79. In some aspects, a hybrid nuclease molecule is as shownin SEQ ID NO: 64, or 79.

In some aspects, a hybrid nuclease molecule comprises a human DNase1G105R A114F domain linked via a (Gly₄Ser)₄ linker domain to a mutant,human IgG1 Fc domain comprising SCC, P238S, and P331S linked via a NLGlinker domain to a wild-type, human RNase1 domain. In some aspects, ahybrid nuclease molecule comprises a human DNase1 G105R A114F domainlinked via a (Gly₄Ser)₄ linker domain to a mutant, human IgG1 Fc domaincomprising SSS, P238S, and P331S, linked via a NLG linker domain to awild-type, human RNase1 domain. In some aspects, a nucleic acid encodinga hybrid nuclease molecule is as shown in SEQ ID NO: 65, or 81. In someaspects, a hybrid nuclease molecule is as shown in SEQ ID NO: 66, or 82.

In other embodiments, a hybrid nuclease molecule comprises an amino acidsequence set forth in SEQ ID NO: 62, 64, 78, 80, 92, or 96, or a hybridnuclease molecule comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO: 62, 64, 78,80, 92, or 96. In some ascpects, a hybrid nuclease molecule comprises anamino acid sequence set forth in SEQ ID NO: 96. In other aspects, ahybrid nuclease molecule comprises an amino acid sequence set forth inSEQ ID NO: 66, 68, 70, 82, 84, 86, 94, or 98, or a hybrid nucleasemolecule comprising an amino acid sequence at least 90% identical to theamino acid sequence set forth in SEQ ID NO: 66, 68, 70, 82, 84, 86, 94,or 98. In other aspects, a hybrid nuclease molecule comprises an aminoacid sequence set forth in SEQ ID NO: 98.

In some aspects, a hybrid nuclease molecule comprises a wild-type, humanRNase1 domain linked via a (Gly₄Ser)₄ linker domain to a mutant, humanIgG1 Fc domain comprising SCC, P238S, and P331S linked via a NLG linkerdomain to a human DNase1 G105R A114F domain. In some aspects, a hybridnuclease molecule comprises a wild-type, human RNase1 domain linked viaa (Gly₄Ser)₄ linker domain to a mutant, human IgG1 Fc domain comprisingSSS, P238S, and P331S, linked via a NLG linker domain to a human DNase1G105R A114F domain. In some aspects, a nucleic acid encoding a hybridnuclease molecule is as shown in SEQ ID NO: 67, or 83. In some aspects,a hybrid nuclease molecule is shown in SEQ ID NO: 68, or 84.

In some aspects, a hybrid nuclease molecule comprises a wild-type, humanRNase1 domain linked to a mutant, human IgG1 Fc domain comprising SCC,P238S, and P331S linked via a NLG linker domain to a human DNase1 G105RA114F domain. In some aspects, a hybrid nuclease molecule comprises awild-type, human RNase1 domain linked to a mutant, human IgG1 Fc domaincomprising SSS, P238S, and P331S linked via a NLG linker domain to ahuman DNase1 G105R A114F domain. In some aspects, a nucleic acidencoding a hybrid nuclease molecule is shown in SEQ ID NO: 69, 85, or93. In some aspects, a hybrid nuclease molecule is shown in SEQ ID NO:70, 86, 94, or 98.

In some aspects, cytotoxicity induced by a hybrid nuclease molecule isreduced when compared to a control molecule. In some aspects,cytotoxicity induced by a hybrid nuclease molecule is reduced about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% when compared to a control molecule. Insome aspects, the hybrid nuclease molecule has about 3-5 fold, or atleast about 3 fold reduced cytotoxicity as compared to a hybrid nucleasemolecule having having an unmodified Fc domain (e.g., a wild type Fcdomain).

In some aspects, the activity of a hybrid nuclease molecule having aDNase is not less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or >30-fold less than the activity of a control DNase molecule. In someaspects, the activity of a hybrid nuclease molecule having a DNase isabout equal to the activity of a control DNase molecule. In someaspects, the activity of a hybrid nuclease molecule having an RNase isnot less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or >30-foldless than the activity of a control RNase molecule. In some aspects, theactivity of a hybrid nuclease molecule having an RNase is about equal tothe activity of a control RNase molecule.

In some embodiments, a hybrid nuclease molecule is a polypeptide,wherein the amino acid sequence of the first nuclease domain comprises ahuman, wild-type RNase amino acid sequence, wherein the first linkerdomain is (Gly4Ser)n, where n is 0, 1, 2, 3, 4 or 5, wherein the aminoacid sequence of the Fc domain comprises a human, mutant IgG1 Fc domainamino acid sequence, and wherein the first linker domain is coupled tothe C-terminus of the first nuclease domain and the N-terminus of the Fcdomain. In some embodiments, a hybrid nuclease molecule is a polypeptidecomprising or consisting of a sequence shown in Table 1.

In some embodiments, a hybrid nuclease molecule comprises wild-type,human DNase1 linked to a mutant, human IgG1 Fc domain. In someembodiments, a hybrid nuclease molecule comprises human DNase1 G105RA114F linked to a mutant, human IgG1 Fc domain by a (Gly₄Ser)_(n) linkerdomain where n=0, 1, 2, 3, 4, or 5. In some embodiments, a hybridnuclease molecule comprises wild-type, human RNase1 linked to a mutant,human IgG1 Fc domain linked to wild-type, human DNase1. In someembodiments, a hybrid nuclease molecule comprises wild-type, humanRNase1 linked to a mutant, human IgG1 Fc domain linked to human DNase1G105R A114F. In some embodiments, a hybrid nuclease molecule is apolypeptide, wherein the amino acid sequence of the first nucleasedomain comprises a RNase amino acid sequence, wherein the first linkerdomain is between 5 and 32 amino acids in length, wherein the amino acidsequence of the Fc domain comprises a human, Fc domain amino acidsequence, and wherein the first linker domain is coupled to theC-terminus of the first nuclease domain and the N-terminus of the Fcdomain. In some embodiments, the linker domain includes (Gly₄Ser)₅ andrestriction sites BglII, AgeI, and XhoI. In some embodiments, a hybridnuclease molecule is a polypeptide, wherein the amino acid sequence ofthe first nuclease domain comprises a human RNase amino acid sequence,wherein the first linker domain is a NLG peptide between 5 and 32 aminoacids in length, wherein the amino acid sequence of the Fc domaincomprises a human, mutant Fc domain amino acid sequence, and wherein thefirst linker domain is coupled to the C-terminus of the first nucleasedomain and the N-terminus of the Fc domain.

In some embodiments, the Fc domain does not substantially bind to an Fcreceptor on a human cell. In some embodiments, the Fc domain is modifiedto decrease binding to Fcγ receptors, complement proteins, or both. Insome aspects Fc receptor binding is reduced about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% when compared to a control molecule. In some aspects,the hybrid nuclease molecule has decrease Fc receptor binding by 3-5fold, or at least about 3 fold as compared to a hybrid nuclease moleculehaving having an unmodified Fc domain (e.g., a wild type Fc domain).

In some embodiments, the serum half-life of the molecule issignificantly longer than the serum half-life of the first nucleasedomain alone. In some embodiments, the nuclease activity of the firstnuclease domain of the molecule is the same or greater than the nucleasedomain alone. In some embodiments, administration of the molecule to amouse increases the survival rate of the mouse as measured by a mouseLupus model assay. In some aspects, the hybrid nuclease moleculedegrades circulating RNA, DNA or both. In other aspects, the hybridnuclease molecule degrades RNA, DNA or both in immune complexes. In someembodiments, hybrid nuclease molecule inhibits interferon-α production.In some aspects interferon-α production is reduced about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% when compared to a control molecule.

In some embodiments, a hybrid nuclease molecule includes a leadersequence. In some embodiments, the leader sequence is human VK3LPpeptide from the human kappa light chain family, and the leader sequenceis coupled to the N-terminus of the first nuclease domain Inembodiments, the VK3LP has the sequence set forth in SEQ ID NO: 100.

In some embodiments, the molecule is a polypeptide. In some embodiments,the molecule is a polynucleotide.

In some embodiments, the first nuclease domain comprises an RNase. Insome embodiments, the RNase is a human RNase. In some embodiments, theRNase is a polypeptide comprising an amino acid sequence at least 90%identical to an RNase amino acid sequence set forth in Table 1. In someembodiments, the RNase is a human RNase A family member. In someembodiments, the RNase is a human pancreatic RNase1.

In some embodiments, the first nuclease domain comprises a DNase. Insome embodiments, the DNase is a human DNase. In some embodiments, theDNase is a polypeptide comprising an amino acid sequence at least 90%identical to a DNase amino acid sequence set forth in Table 1. In someembodiments, the DNase is selected from the group consisting of humanDNase I, TREX1, and human DNase 1L3.

In some embodiments, the Fc domain is a human Fc domain. In someembodiments, the Fc domain is a mutant Fc domain. In some embodiments,the Fc domain is a mutant Fc domain comprising SSS, P238S, and/or P331S.In some embodiments, the Fc domain is a human IgG1 Fc domain. In someembodiments, the Fc domain is a polypeptide comprising an amino acidsequence at least 90% identical to an Fc domain amino acid sequence setforth in Table 1.

In some embodiments, the first linker domain has a length of about 1 toabout 50 amino acids. In some embodiments, the first linker domain has alength of about 5 to about 31 amino acids. In some embodiments, thefirst linker domain has a length of about 15 to about 25 amino acids. Insome embodiments, the first linker domain has a length of about 20 toabout 32 amino acids. In some embodiments, the first linker domain has alength of about 20 amino acids. In some embodiments, the first linkerdomain has a length of about 25 amino acids. In some embodiments, thefirst linker domain has a length of about 18 amino acids. In someembodiments, the first linker domain comprises a gly/ser peptide. Insome embodiments, the gly/ser peptide is of the formula (Gly₄Ser)_(n),wherein n is a positive integer selected from the group consisting of 1,2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the gly/ser peptideincludes (Gly₄Ser)₃. In some embodiments, the gly/ser peptide includes(Gly₄Ser)₄. In some embodiments, the gly/ser peptide includes(Gly₄Ser)₅. In some embodiments, the first linker domain includes atleast one restriction site. In some embodiments, the first linker domainincludes about 12 or greater nucleotides including at least onerestriction site. In some embodiments, the first linker domain includestwo or more restriction sites. In some embodiments, the first linkerdomain includes a plurality of restriction sites. In some embodiments,the first linker domain comprises an NLG peptide. NLG peptides containan N-linked glycosylation consensus sequence. In embodiments, the NLGpeptide has a sequence as set forth in SEQ ID NO: 99. In someembodiments, the first linker domain comprises an N-linked glycosylationsite.

In some embodiments, the first nuclease domain is linked to theN-terminus of the Fc domain In some embodiments, the first nucleasedomain is linked to the C-terminus of the Fc domain.

In some embodiments, the hybrid nuclease molecule further includes asecond nuclease domain. In some embodiments, the first and secondnuclease domains are distinct nuclease domains. In some embodiments, thefirst and second nuclease domains are the same nuclease domains. In someembodiments, the second nuclease domain is linked to the C-terminus ofthe Fc domain In some embodiments, the second nuclease domain is linkedto the N-terminus of the Fc domain. In some embodiments, the secondnuclease domain is linked to the C-terminus of the first nucleasedomain. In some embodiments, the second nuclease domain is linked to theN-terminus of the first nuclease domain.

Also disclosed herein is a dimeric polypeptide comprising a firstpolypeptide and a second polypeptide, wherein the first polypeptidecomprises a first nuclease domain, and an Fc domain, wherein the firstnuclease domain is operatively coupled to the Fc domain. In someembodiments, the second polypeptide is a second hybrid nuclease moleculecomprising a second nuclease domain, and a second Fc domain, wherein thesecond nuclease domain is operatively coupled to the second Fc domain.

Also disclosed herein is a pharmaceutical composition comprising atleast one hybrid nuclease molecule and/or at least one dimericpolypeptide as described herein, and a pharmaceutically acceptableexcipient.

Also disclosed herein is a nucleic acid molecule encoding a hybridnuclease molecule disclosed herein. Also disclosed herein is arecombinant expression vector comprising a nucleic acid moleculedisclosed herein. Also disclosed herein is a host cell transformed witha recombinant expression vector disclosed herein.

Also disclosed herein is a method of making a hybrid nuclease disclosedherein, comprising: providing a host cell comprising a nucleic acidsequence that encodes the hybrid nuclease molecule; and maintaining thehost cell under conditions in which the hybrid nuclease molecule isexpressed.

Also disclosed herein is a method for treating or preventing a conditionassociated with an abnormal immune response, comprising administering toa patient in need thereof an effective amount of an isolated hybridnuclease molecule disclosed herein. In some embodiments, the conditionis an autoimmune disease. In some embodiments, the autoimmune disease isselected from the group consisting of insulin-dependent diabetesmellitus, multiple sclerosis, experimental autoimmune encephalomyelitis,rheumatoid arthritis, experimental autoimmune arthritis, myastheniagravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto'sthyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia,autoimmune atrophic gastritis, Addison's disease, premature menopause,male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigusvulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis,autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliarycirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis,ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener'sgranulomatosis, polymyositis, dermatomyositis, discoid LE, systemiclupus erythematosus (SLE), and connective tissue disease. In someembodiments, the autoimmune disease is SLE.

Also disclosed herein is a method of treating SLE comprisingadministering to a subject a nuclease-containing composition in anamount effective to degrade immune complexes containing RNA, DNA or bothRNA and DNA. In some aspects, the composition includes apharmaceutically acceptable carrier and a hybrid nuclease molecule asdescribed herein. In other aspects, the composition includes a hybridnuclease molecule comprising an amino acid sequence set forth in SEQ IDNO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows a prototype structure for creating different embodiments ofhybrid nuclease molecules.

FIG. 2 shows the concentration of RSLV-124 recovered from mouse serumfollowing a single intravenous injection.

FIG. 3 shows results of an RNase enzymatic activity assay on RLSV-124recovered from mouse serum as measured in relative fluorescence units(RFU's) over time.

FIG. 4 shows the concentration of RSLV-124 in mouse serum asextrapolated from the RNase enzymatic activity of the molecule.

FIG. 5 shows single radial enzyme diffusion (SRED) analysis of serumfrom two RNase transgenic (Tg) mice compared to a normal B6 mouse.

FIG. 6 shows the concentration of RNaseA in Tg and double Tg (DTg) micemeasured by ELISA. Each dot represents the concentration measured in anindividual mouse.

FIG. 7 shows survival of TLR7.1 Tg versus TLR7.1xRNaseA DTg mice

FIG. 8 shows quantitative PCR of IRGs in spleens of Tg versus DTg mice.

FIG. 9 shows a Western Blot on COS transfection supernatants from RSLV125-129 constructs (SEQ ID NOs 208-217).

FIG. 10 shows SRED analysis comparing aliquots of protein A purifiedproteins from RSLV transfected COS supernatants.

FIG. 11A shows results from a DNase nuclease activity assay performed onprotein A purified protein from COS7 supernatants transfected with RSLVfusion plasmids.

FIG. 11B shows results from a DNase nuclease activity assay performed onprotein A purified protein from COS7 supernatants transfected with RSLVfusion plasmids.

FIG. 11C shows results from a DNase nuclease activity assay performed onprotein A purified protein from COS7 supernatants transfected with RSLVfusion plasmids.

FIG. 12 shows the RFU (relative fluorescence units) as a function oftime for each protein.

FIG. 13 shows a Lineweaver Burk plot of the different molecules tested.

FIG. 14 shows cytotoxicity data graphing the percentage of dead cells asa function of concentration of fusion protein for RNaseIg molecules witha wild type or mutant Fc domain.

FIG. 15 shows histogram overlays of THP-1 stained cells after 72 hours.

FIG. 16 shows the ability of RSLV-132 to inhibit interferon-α productioninduced by SLE patient immune complexes.

FIG. 17 shows the in vivo ability of RSLV-132 to inhibit RNA-inducedinterferon-α production.

FIG. 18 shows an RNase enzymatic activity assay of two production lotsof RSLV-132 having been stored for up to 8 weeks at 4C, compared to wildtype RNase and RSLV-124.

FIG. 19 shows an RNase enzymatic activity assay of RSLV-133, RSLV-123and RSLV-124 relative to RNase A as measured in RFUs over time.

FIG. 20 shows a DNase enzymatic activity assay of RSLV-133 and RSLV-123relative to DNase 1 as measured in RFUs over time.

FIG. 21 shows the results of an in-gel digestion experiment comparingthe ability of RSLV-133 to digest DNA relative to RSLV-123 and wild typeDNase 1.

FIG. 22 shows binding of RSLV-124 and RSLV-132 to Fc-receptor bearingTHP1 cells by FACS analysis measuring mean fluorescence intensity.

DETAILED DESCRIPTION

Systemic lupus erythematosus (SLE) is a multisystem autoimmune diseasecharacterized by the presence of high titer autoantibodies directedagainst self nucleoproteins. There is strong evidence that defectiveclearance or processing of dead and dying cells in SLE leads to disease,predominantly through accumulation of ribo- and deoxy-ribonucleoproteins(abbreviated nucleoproteins). The nucleoproteins cause damage throughthree mechanisms: i) activation of the innate immune system to produceinflammatory cytokines; ii) serve as antigens to generate circulatingimmune complexes; and iii) serve as antigens to generate in situ complexformation at local sites such as the kidney. The present invention isbased, at least in part, on the discovery that digestion of theextracellular nucleic acids has a therapeutic effect in vivo.

Accordingly, the present invention provides methods for treatingdiseases characterized by defective clearance or processing of apoptoticcells and cell debris, such as SLE, by administering an effective amountof a nuclease activity to degrade extracellular RNA and DNA containingcomplexes. Such treatment can inhibit production of Type I interferons(IFNs) which are prominent cytokines in SLE and are strongly correlatedwith disease activity and nephritis.

In one embodiment, a subject is treated by administering a nucleaseactivity which is a DNase or an RNase activity, preferably in the formof a hybrid nuclease molecule. In one aspect, the nuclease activity is afirst nuclease domain In another aspect, the nuclease domain is coupledto a modified Fc domain such that the molecule has reduced cytotoxicity.In one aspect a hybrid nuclease molecule includes a second nucleasedomain

In another aspect, a method of treating SLE is provided in which aneffective amount of a nuclease-containing composition is administered toa subject. In one aspect, treatment results in degradation of immunecomplexes containing RNA, DNA or both RNA and DNA. In another aspect,treatment results in inhibition of Type I interferons, such asinterferon-α in a subject. In one aspect, a method of treating a subjectcomprises administering an effective amount of a composition of a hybridnuclease molecule comprising an amino acid sequence set forth in SEQ IDNO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98. Inanother aspect, the composition is a hybrid nuclease molecule comprisingan amino acid sequence set forth in SEQ ID NO: 96 or 98.

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. In the case of direct conflict with aterm used in a parent provisional patent application, the term used inthe instant specification shall control.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, the presentlarger “peptide insertions,” can be made, e.g. insertion of about threeto about five or even up to about ten, fifteen, or twenty amino acidresidues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

“Polypeptide,” “peptide”, and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991;Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985); and Cassol et al.,1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). For arginineand leucine, modifications at the second base can also be conservative.The term nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene.

Polynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

As used herein, the term “hybrid nuclease molecule” refers topolynucleotides or polypeptides that comprise at least one nucleasedomain and at least one Fc domain Hybrid nuclease molecules are alsoreferred to as fusion protein(s) and fusion gene(s). For example, in oneembodiment, a hybrid nuclease molecule can be a polypeptide comprisingat least one Fc domain linked to a nuclease domain such as DNase and/orRNase. As another example, a hybrid nuclease molecule can include anRNase nuclease domain, a linker domain, and an Fc domain Examples ofhybrid nuclease molecules include SE ID NO:62, 64, 66, 68, 70, 78, 80,82, 84, 86, 92, 94, 96, and 98. Other examples are described in moredetail below. In one embodiment a hybrid nuclease molecule of theinvention can include additional modifications. In another embodiment, ahybrid nuclease molecule may be modified to add a functional moiety(e.g., PEG, a drug, or a label).

As used herein, a “hybrid bispecific nuclease molecule,” or a“binuclease molecule” refer to a hybrid nuclease molecule with 2 or morenuclease domains, e.g., a DNase domain and an RNase domain.

In certain aspects, the hybrid nuclease molecules of the invention canemploy one or more “linker domains,” such as polypeptide linkers. Asused herein, the term “linker domain” refers to a sequence whichconnects two or more domains in a linear sequence. As used herein, theterm “polypeptide linker” refers to a peptide or polypeptide sequence(e.g., a synthetic peptide or polypeptide sequence) which connects twoor more domains in a linear amino acid sequence of a polypeptide chain.For example, polypeptide linkers may be used to connect a nucleasedomain to an Fc domain. Preferably, such polypeptide linkers can provideflexibility to the polypeptide molecule. In certain embodiments thepolypeptide linker is used to connect (e.g., genetically fuse) one ormore Fc domains and/or one or more nuclease domains. A hybrid nucleasemolecule of the invention may comprise more than one linker domain orpeptide linker.

As used herein, the term “gly-ser polypeptide linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser polypeptide linker comprises the amino acid sequenceSer(Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2. Inanother embodiment, n=3, i.e., Ser(Gly₄Ser)3. In another embodiment,n=4, i.e., Ser(Gly₄Ser)4. In another embodiment, n=5. In yet anotherembodiment, n=6. In another embodiment, n=7. In yet another embodiment,n=8. In another embodiment, n=9. In yet another embodiment, n=10.Another exemplary gly/ser polypeptide linker comprises the amino acidsequence Ser(Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2.In a preferred embodiment, n=3. In another embodiment, n=4. In anotherembodiment, n=5. In yet another embodiment, n=6.

As used herein, the terms “linked,” “fused”, or “fusion”, are usedinterchangeably. These terms refer to the joining together of two moreelements or components or domains, by whatever means including chemicalconjugation or recombinant means. Methods of chemical conjugation (e.g.,using heterobifunctional crosslinking agents) are known in the art.

As used herein, the term “Fc region” shall be defined as the portion ofa native immunoglobulin formed by the respective Fc domains (or Fcmoieties) of its two heavy chains.

As used herein, the term “Fc domain” refers to a portion of a singleimmunoglobulin (Ig) heavy chain wherein the Fc domain does not comprisean Fv domain. As such, Fc domain can also be referred to as “Ig” or“IgG.” In some embodiments, an Fc domain begins in the hinge region justupstream of the papain cleavage site and ending at the C-terminus of theantibody. Accordingly, a complete Fc domain comprises at least a hingedomain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fcdomain comprises at least one of: a hinge (e.g., upper, middle, and/orlower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, ora variant, portion, or fragment thereof. In other embodiments, an Fcdomain comprises a complete Fc domain (i.e., a hinge domain, a CH2domain, and a CH3 domain) In one embodiment, an Fc domain comprises ahinge domain (or portion thereof) fused to a CH3 domain (or portionthereof). In another embodiment, an Fc domain comprises a CH2 domain (orportion thereof) fused to a CH3 domain (or portion thereof). In anotherembodiment, an Fc domain consists of a CH3 domain or portion thereof. Inanother embodiment, an Fc domain consists of a hinge domain (or portionthereof) and a CH3 domain (or portion thereof). In another embodiment,an Fc domain consists of a CH2 domain (or portion thereof) and a CH3domain. In another embodiment, an Fc domain consists of a hinge domain(or portion thereof) and a CH2 domain (or portion thereof). In oneembodiment, an Fc domain lacks at least a portion of a CH2 domain (e.g.,all or part of a CH2 domain). In one embodiment, an Fc domain of theinvention comprises at least the portion of an Fc molecule known in theart to be required for FcRn binding. In another embodiment, an Fc domainof the invention comprises at least the portion of an Fc molecule knownin the art to be required for FcγR binding. In one embodiment, an Fcdomain of the invention comprises at least the portion of an Fc moleculeknown in the art to be required for Protein A binding. In oneembodiment, an Fc domain of the invention comprises at least the portionof an Fc molecule known in the art to be required for protein G binding.An Fc domain herein generally refers to a polypeptide comprising all orpart of the Fc domain of an immunoglobulin heavy-chain. This includes,but is not limited to, polypeptides comprising the entire CH1, hinge,CH2, and/or CH3 domains as well as fragments of such peptides comprisingonly, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derivedfrom an immunoglobulin of any species and/or any subtype, including, butnot limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody. The Fc domain encompasses native Fc and Fc variant molecules.As with Fc variants and native Fc's, the term Fc domain includesmolecules in monomeric or multimeric form, whether digested from wholeantibody or produced by other means. The assignment of amino acidsresidue numbers to an Fc domain is in accordance with the definitions ofKabat. See, e.g., Sequences of Proteins of Immunological Interest (Tableof Contents, Introduction and Constant Region Sequences sections), 5thedition, Bethesda, Md.:NIH vol. 1:647-723 (1991); Kabat et al.,“Introduction” Sequences of Proteins of Immunological Interest, US Deptof Health and Human Services, NIH, 5th edition, Bethesda, Md. vol.1:xiii-xcvi (1991); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989), each of which is hereinincorporated by reference for all purposes.

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain exemplary embodiments, the Fc domainretains an effector function (e.g., FcγR binding).

The Fc domains of a polypeptide of the invention may be derived fromdifferent immunoglobulin molecules. For example, an Fc domain of apolypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, an Fc domain can comprise a chimeric hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, an Fc domain can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence.

Polypeptides derived from another peptide may have one or more mutationsrelative to the starting polypeptide, e.g., one or more amino acidresidues which have been substituted with another amino acid residue orwhich has one or more amino acid residue insertions or deletions.

A polypeptide can comprise an amino acid sequence which is not naturallyoccurring. Such variants necessarily have less than 100% sequenceidentity or similarity with the starting hybrid nuclease molecules. In apreferred embodiment, the variant will have an amino acid sequence fromabout 75% to less than 100% amino acid sequence identity or similaritywith the amino acid sequence of the starting polypeptide, morepreferably from about 80% to less than 100%, more preferably from about85% to less than 100%, more preferably from about 90% to less than 100%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferablyfrom about 95% to less than 100%, e.g., over the length of the variantmolecule.

In one embodiment, there is one amino acid difference between a startingpolypeptide sequence and the sequence derived therefrom. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

In one embodiment, a polypeptide of the invention consists of, consistsessentially of, or comprises an amino acid sequence selected from Table1 and functionally active variants thereof. In an embodiment, apolypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth in Table 1. Inan embodiment, a polypeptide includes a contiguous amino acid sequenceat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguousamino acid sequence set forth in Table 1. In an embodiment, apolypeptide includes an amino acid sequence having at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200,300, 400, or 500 (or any integer within these numbers) contiguous aminoacids of an amino acid sequence set forth in Table 1.

In an embodiment, the peptides of the invention are encoded by anucleotide sequence. Nucleotide sequences of the invention can be usefulfor a number of applications, including: cloning, gene therapy, proteinexpression and purification, mutation introduction, DNA vaccination of ahost in need therof, antibody generation for, e.g., passiveimmunization, PCR, primer and probe generation, siRNA design andgeneration (see, e.g., the Dharmacon siDesign website), and the like. Inan embodiment, the nucleotide sequence of the invention comprises,consists of, or consists essentially of, a nucleotide sequence selectedfrom Table 1. In an embodiment, a nucleotide sequence includes anucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a nucleotide sequence set forth in Table 1. In an embodiment, anucleotide sequence includes a contiguous nucleotide sequence at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotidesequence set forth in Table 1. In an embodiment, a nucleotide sequenceincludes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or500 (or any integer within these numbers) contiguous nucleotides of anucleotide sequence set forth in Table 1.

Preferred hybrid nuclease molecules of the invention comprise a sequence(e.g., at least one Fc domain) derived from a human immunoglobulinsequence. However, sequences may comprise one or more sequences fromanother mammalian species. For example, a primate Fc domain or nucleasedomain may be included in the subject sequence. Alternatively, one ormore murine amino acids may be present in a polypeptide. In someembodiments, polypeptide sequences of the invention are not immunogenicand/or have reduced immunogenicity.

It will also be understood by one of ordinary skill in the art that thehybrid nuclease molecules of the invention may be altered such that theyvary in sequence from the naturally occurring or native sequences fromwhich they were derived, while retaining the desirable activity of thenative sequences. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at “non-essential”amino acid residues may be made. An isolated nucleic acid moleculeencoding a non-natural variant of a hybrid nuclease molecule derivedfrom an immunoglobulin (e.g., an Fc domain) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.

The peptide hybrid nuclease molecules of the invention may compriseconservative amino acid substitutions at one or more amino acidresidues, e.g., at essential or non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in a binding polypeptide is preferably replaced with anotheramino acid residue from the same side chain family In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members. Alternatively, in another embodiment, mutations may beintroduced randomly along all or part of a coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be incorporatedinto binding polypeptides of the invention and screened for theirability to bind to the desired target.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., an autoimmune disease state(e.g., SLE), including prophylaxis, lessening in the severity orprogression, remission, or cure thereof.

The term “in situ” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and include but is not limited to humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to modulate proteinaggregation in a cell.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions

Hybrid Nuclease Molecules

In some embodiments, a composition of the invention includes a hybridnuclease molecule. In some embodiments, a hybrid nuclease moleculeincludes a nuclease domain operatively linked to an Fc domain. In someembodiments, a hybrid nuclease molecule includes a nuclease domainlinked to an Fc domain. In some embodiments the hybrid nuclease moleculeis a nuclease protein. In some embodiments, the hybrid nuclease moleculeis a nuclease polynucleotide.

In some embodiments, the nuclease domain is linked to the Fc domain viaa linker domain In some embodiments, the linker domain is a linkerpeptide. In some embodiments, the linker domain is a linker nucleotide.In some embodiments, the hybrid nuclease molecule includes a leadermolecule, e.g., a leader peptide. In some embodiments, the leadermolecule is a leader peptide positioned at the N-terminus of thenuclease domain. In embodiments, a hybrid nuclease molecule of theinvention comprises a leader peptide at the N-terminus of the molecule,wherein the leader peptide is later cleaved from the hybrid nucleasemolecule. Methods for generating nucleic acid sequences encoding aleader peptide fused to a recombinant protein are well known in the art.In embodiments, any of the hybrid nuclease molecules of the presentinvention can be expressed either with or without a leader fused totheir N-terminus. The protein sequence of a hybrid nuclease molecule ofthe present invention following cleavage of a fused leader peptide canbe predicted and/or deduced by one of skill in the art. Examples ofhybrid nuclease molecules of the present invention additionallyincluding a VK3 leader peptide (VK3LP), wherein the leader peptide isfused to the N-terminus of the hybrid nuclease molecule, are set forthin SEQ ID NOS: 92 (RSLV-132) and 94 (RSLV-133). The correspondingnucleotide sequences are set forth in SEQ ID NOS: 91 and 93,respectively. In embodiments, following cleavage of the VK3 leader,these hybrid nuclease molecules have the sequences as set forth in SEQID NOS: 96 (RSLV-132) and 98 (RSLV-133), respectively. The correspondingnucleotide sequences are set forth in SEQ ID NOS: 95 and 97,respectively. In some embodiments, a hybrid nuclease molecule of thepresent invention is expressed without a leader peptide fused to itsN-terminus, and the resulting hybrid nuclease molecule has an N-terminalmethionine.

In some embodiments, the hybrid nuclease molecule will include a stopcodon. In some embodiments, the stop codon will be at the C-terminus ofthe Fc domain.

In some embodiments, the hybrid nuclease molecule further includes asecond nuclease domain. In some embodiments, the second nuclease domainis linked to the Fc domain via a second linker domain. In someembodiments, the second linker domain will be at the C-terminus of theFc domain FIG. 1 shows at least one embodiment of a hybrid nucleasemolecule. In some embodiments, a hybrid nuclease molecule includes asequence shown in Table 1.

In some embodiments, a hybrid nuclease molecule is an RNase molecule orDNase molecule or a multi-enzyme molecule (e.g., both RNase and DNase ortwo RNA or DNA nucleases with different specificity for substrate)attached to an Fc domain that specifically binds to extracellular immunecomplexes. In some embodiments, the Fc domain does not effectively bindFcγ receptors. In one aspect, the hybrid nuclease molecule does noteffectively bind C1q. In other aspects, the hybrid nuclease moleculecomprises an in frame Fc domain from IgG1. In other aspects, the hybridnuclease molecule further comprises mutations in the hinge, CH2, and/orCH3 domains. In other aspects, the mutations are P238S, P331S or N297S,and may include mutations in one or more of the three hinge cysteines.In some such aspects, the mutations in one or more of three hingecysteines can be SCC or SSS. In other aspects, the molecules contain theSCC hinge, but are otherwise wild type for human IgG1 Fc CH2 and CH3domains, and bind efficiently to Fc receptors, facilitating uptake ofthe hybrid nuclease molecule into the endocytic compartment of cells towhich they are bound. In other aspects, the molecule has activityagainst single and/or double-stranded RNA substrates.

In some aspects, a hybrid nuclease molecule includes a mutant Fc domain.In some aspects, a hybrid nuclease molecule includes a mutant, IgG1 Fcdomain In some aspects, a mutant Fc domain comprises one or moremutations in the hinge, CH2, and/or CH3 domains. In some aspects, amutant Fc domain includes a P238S mutation. In some aspects, a mutant Fcdomain includes a P331S mutation. In some aspects, a mutant Fc domainincludes a P238S mutation and a P331S mutation. In some aspects, amutant Fc domain comprises P238S and/or P331S, and may include mutationsin one or more of the three hinge cysteines. In some aspects, a mutantFc domain comprises P238S and/or P331S, and/or one or more mutations inthe three hinge cysteines. In some aspects, a mutant Fc domain comprisesP238S and/or P331S, and/or mutations in the three hinge cysteines to SSSor in one hinge cysteine to SCC. In some aspects, a mutant Fc domaincomprises P238S and P331S and mutations in the three hinge cysteines. Insome aspects, a mutant Fc domain comprises P238S and P331S and eitherSCC or SSS. In some aspects, a mutant Fc domain comprises P238S andP331S and SCC. In some aspects, a mutant Fc domain includes P238S SSS.In some aspects, a mutant Fc domain includes P331S and either SCC orSSS. In some aspects, a mutant Fc domain includes mutations in one ormore of the three hinge cysteines. In some aspects, a mutant Fc domainincludes mutations in the three hinge cysteines. In some aspects, amutant Fc domain includes mutations in the three hinge cysteines to SSS.In some aspects, a mutant Fc domain includes mutations in one of thethree hinge cysteines to SCC. In some aspects, a mutant Fc domainincludes SCC or SSS. In some aspects, a mutant Fc domain is as shown inany of SEQ ID NOs 59, 60, 71-76, or 87-90. In some aspects, a hybridnuclease molecule is as shown in any of SEQ ID NOs 62, 64, 66, 68, 70,78, 80, 82, 84, 86, 92, 94, 96, or 98. In some aspects, a hybridnuclease molecule comprises a wild-type, human RNase1 domain linked to amutant, human IgG1 Fc domain comprising SCC, P238S, and P331S, or amutant, human IgG1 Fc domain comprising SSS, P238S, and P331S. In someaspects, a nucleic acid sequence encoding a hybrid nuclease molecule isas shown in SEQ ID NO: 61, 77, or 91. In some aspects, a hybrid nucleasemolecule is shown in SEQ ID NO: 62, 78, 92, or 96.

In some aspects, a hybrid nuclease molecule comprises a wild-type, humanRNase1 domain linked via a (Gly4Ser)4 linker domain to a mutant, humanIgG1 Fc domain comprising SCC, P238S, and P331S or a mutant, human IgG1Fc domain comprising SSS, P238S, and P331S. In some aspects, a nucleicacid sequence encoding a hybrid nuclease molecule is shown in SEQ ID NO:63, or 79. In some aspects, a hybrid nuclease molecule is shown in SEQID NO: 64, or 80.

In some aspects, a hybrid nuclease molecule comprises a human DNase1G105R A114F domain linked via a (Gly₄Ser)4 linker domain to a mutant,human IgG1 Fc domain comprising SCC, P238S, and P331S linked via a NLGlinker domain to a wild-type, human RNase1 domain. In some aspects, ahybrid nuclease molecule comprises a human DNase1 G105R A114F domainlinked via a (Gly₄Ser)4 linker domain to a mutant, human IgG1 Fc domaincomprising SSS, P238S, and P331S linked via a NLG linker domain to awild-type, human RNase1 domain. In some aspects, a nucleic acid sequenceencoding a hybrid nuclease molecule is shown in SEQ ID NO: 65, or 81. Insome aspects, a hybrid nuclease molecule is shown in SEQ ID NO: 66, or82.

In some aspects, a hybrid nuclease molecule comprises a wild-type, humanRNase1 domain linked via a (Gly4Ser)4 linker domain to a mutant, humanIgG1 Fc domain comprising SCC, P238S, and P331S linked via a NLG linkerdomain to a human DNase1 G105R A114F domain. In some aspects, a hybridnuclease molecule comprises a wild-type, human RNase1 domain linked viaa (Gly₄Ser)4 linker domain to a mutant, human IgG1 Fc domain comprisingSSS, P238S, and P331S linked via a NLG linker domain to a human DNase1G105R A114F domain In some aspects, a nucleic acid sequence encoding ahybrid nuclease molecule is shown in SEQ ID NO: 67, or 83. In someaspects, a hybrid nuclease molecule is shown in SEQ ID NO: 68, or 84.

In some aspects, a hybrid nuclease molecule comprises a wild-type, humanRNase1 domain linked to a mutant, human IgG1 Fc domain comprising SCC,P238S, and P331S linked via a NLG linker domain to a human DNase1 G105RA114F domain. In some aspects, a hybrid nuclease molecule comprises awild-type, human RNase1 domain linked to a mutant, human IgG1 Fc domaincomprising SSS, P238S, and P331S linked via a NLG linker domain to ahuman DNase1 G105R A114F domain. In some aspects, a nucleic acidsequence encoding a hybrid nuclease molecule is shown in SEQ ID NO: 69,85, or 93. In some aspects, a hybrid nuclease molecule is shown in SEQID NO: 70, 86, 94, or 98.

In some aspects, the activity of the hybrid nuclease molecule isdetectable in vitro and/or in vivo. In some aspects, the hybrid nucleasemolecule binds to a cell, a malignant cell, or a cancer cell andinterferes with its biologic activity.

In another aspect, a multifunctional RNase molecule is provided that isattached to another enzyme or antibody having binding specificity, suchas an scFv targeted to RNA or a second nuclease domain with the same ordifferent specificities as the first domain.

In another aspect, a multifunctional DNase molecule is provided that isattached to another enzyme or antibody having binding specificity, suchas an scFv targeted to DNA or a second nuclease domain with the same ordifferent specificities as the first domain.

In another aspect, a hybrid nuclease molecule is adapted for preventingor treating a disease or disorder in a mammal by administering an hybridnuclease molecule attached to an Fc region, in a therapeuticallyeffective amount to the mammal in need thereof, wherein the disease isprevented or treated. In other aspects, the disease or disorder is anautoimmune disease or cancer. In some such aspects, the autoimmunedisease is insulin-dependent diabetes mellitus, multiple sclerosis,experimental autoimmune encephalomyelitis, rheumatoid arthritis,experimental autoimmune arthritis, myasthenia gravis, thyroiditis, anexperimental form of uveoretinitis, Hashimoto's thyroiditis, primarymyxoedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophicgastritis, Addison's disease, premature menopause, male infertility,juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris,pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmunehaemolytic anaemia, idiopathic leucopenia, primary biliary cirrhosis,active chronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerativecolitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis,polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus,or connective tissue disease.

In some embodiments, the targets of the RNase enzyme activity of RNasehybrid nuclease molecules are primarily extracellular, consisting of,e.g., RNA contained in immune complexes with anti-RNP autoantibody andRNA expressed on the surface of cells undergoing apoptosis. In someembodiments, the RNase hybrid nuclease molecule is active in the acidicenvironment of the endocytic vesicles. In some embodiments, an RNasehybrid nuclease molecule includes a wild-type (wt) Fc domain in orderto, e.g, allow the molecule to bind FcR and enter the endocyticcompartment through the entry pathway used by immune complexes. In someembodiments, an RNase hybrid nuclease molecule including a wt Fc domainis adapted to be active both extracellularly and in the endocyticenvironment (where TLR7 can be expressed). In some aspects, this allowsan RNase hybrid nuclease molecule including a wt Fc domain to stop TLR7signaling through previously engulfed immune complexes or by RNAs thatactivate TLR7 after viral infection. In some embodiments, the wt RNaseof an RNase hybrid nuclease molecule is not resistant to inhibition byan RNase cytoplasmic inhibitor. In some embodiments, the wt RNase of anRNase hybrid nuclease molecule is not active in the cytoplasm of a cell.

In some embodiments, a hybrid nuclease molecule including a wt Fc domainis used for therapy of an autoimmune disease, e.g., SLE.

In some embodiments, Fc domain binding to an Fc receptor (FcR) isincreased, e.g., via alterations of glycosylation and/or changes inamino acid sequence. In some embodiments, a hybrid nuclease molecule hasone or more Fc alterations that increase FcR binding.

Alternative ways to construct a hybrid nuclease molecule attached to anFc domain are envisioned. In some embodiments, the domain orientationcan be altered to construct an Ig-RNase molecule or an Ig-DNase moleculeor an RNase-Ig molecule or an RNase-Ig molecule that retains FcR bindingand has active nuclease domains.

In some embodiments, DNase hybrid nuclease molecules include a wt Fcdomain that can allow, e.g., the molecules to undergo endocytosis afterbinding FcR. In some embodiments, the DNase hybrid nuclease moleculescan be active towards extracellular immune complexes containing DNA,e.g., either in soluble form or deposited as insoluble complexes.

In some embodiments, hybrid nuclease molecules include both DNase andRNase. In some embodiments, these hybrid nuclease molecules can improvethe therapy of SLE because they can, e.g., digest immune complexescontaining RNA, DNA, or a combination of both RNA and DNA; and when theyfurther include a wt Fc domain, they are active both extracellularly andin the endocytic compartment where TLR7 and TLR9 can be located.

In some embodiments, linker domains include (gly4ser) 3, 4 or 5 variantsthat alter the length of the linker by 5 amino acid progressions. Inanother embodiment, a linker domain is approximately 18 amino acids inlength and includes an N-linked glycosylation site, which can besensitive to protease cleavage in vivo. In some embodiments, an N-linkedglycosylation site can protect the hybrid nuclease molecules fromcleavage in the linker domain. In some embodiments, an N-linkedglycosylation site can assist in separating the folding of independentfunctional domains separated by the linker domain.

In some embodiments, hybrid nuclease molecules can include both mutantand/or wild type human IgG1 Fc domains. In some embodiments, the hybridnuclease molecules can be expressed from both COS transient and CHOstable transfections. In some embodiments, both the CD80/86 binding andthe RNase activity are preserved in a hybrid nuclease molecule. In someembodiments, hybrid nuclease molecules include DNase1L3-Ig-linker-RNaseconstructs. In some embodiments, a hybrid nuclease molecule includes aDNase1-Ig-linker-RNase construct or an RNase-Ig-linker-DNase construct.In some embodiments, fusion junctions between enzyme domains and theother domains of the hybrid nuclease molecule is optimized.

In some embodiments, hybrid nuclease molecules include DNase-Ig hybridnuclease molecules and/or hybrid DNase-RNase hybrid nuclease molecules.

In some embodiments, a hybrid nuclease molecule includes TREX1. In someembodiments, a TREX1 hybrid nuclease molecule can digest chromatin. Insome embodiments, a TREX1 hybrid nuclease molecule is expressed by acell. In some embodiments, the expressed hybrid nuclease moleculeincludes murine TREX-1 and a murine (wt or mutant) Fc domain. In someembodiments, a 20-25 amino acid (aa) linker domain between TREX1 and theIgG hinge can be required to allow DNase activity. In some embodiments,a hybrid nuclease molecule with a 15 aa linker domain is not active. Insome embodiments, use of the 20 and 25 amino acid linker domains (plus 2or more amino acids to incorporate restriction sites) results infunctional activity as measured by chromatin digestion. In someembodiments, a hydrophobic region of approximately 72 aa can be removedfrom the COOH end of TREX-1 prior to fusion to the Fc domain via thelinker domain. In some embodiments, a 20 amino acid linker domainversion of the hybrid nuclease molecule exhibits high expression levelscompared to controls and/or other hybrid nuclease molecules. In someembodiments, kinetic enzyme assays are used to compare the enzymeactivity of hybrid nuclease molecules and controls in a quantitativemanner

In some embodiments, further optimization of the fusion junction chosenfor truncation of a TREX1 enzyme can be used to improve expression ofthe hybrid nuclease molecules.

In some embodiments, the hybrid nuclease molecule includes a humanTREX1-linker-Ig Fc domain hybrid nuclease molecule with 20 and/or 25 aalinker domains. In some embodiments, the linker domain(s) are variantsof a (gly4ser)4 or (gly4ser)5 cassette with one or more restrictionsites attached for incorporation into the hybrid nuclease moleculesconstruct. In some embodiments, because of the head-to-tail dimerizationuseful for TREX1 enzyme activity; a flexible, longer linker domain canbe used to facilitate proper folding.

In some embodiments, the hybrid nuclease molecule is a TREX1-tandemhybrid nuclease molecule. In some embodiments, an alternative method forfacilitating head-to-tail folding of TREX1 is to generate aTREX1-TREX1-Ig hybrid hybrid nuclease molecule that incorporates twoTREX1 domains in tandem, followed by a linker domain and an Ig Fcdomain. In some embodiments, positioning of TREX1 cassettes in ahead-to-tail manner can be corrected for head-to tail folding on eitherarm of the immunoenzyme and introduce a single TREX1 functional domaininto each arm of the molecule. In some embodiments, each immunoenzyme ofa hybrid nuclease molecule has two functional TREX1 enzymes attached toa single IgG Fc domain.

In some embodiments, the hybrid nuclease molecule includesTREX1-linker1-Ig-linker2-RNase.

In some embodiments, the hybrid nuclease molecule includesRNase-Ig-linker-TREX1. In some embodiments, cassettes are derived forboth amino and carboxyl fusion of each enzyme for incorporation intohybrid nuclease molecules where the enzyme configuration is reversed. Insome embodiments, the RNase enzyme exhibits comparable functionalactivity regardless of its position in the hybrid nuclease molecules. Insome embodiments, alternative hybrid nuclease molecules can be designedto test whether a particular configuration demonstrates improvedexpression and/or function of the hybrid nuclease molecule components.

In some embodiments, the hybrid nuclease molecule includes 1L3-Ig. Insome embodiments, the 1L3 DNase is constructed from a murine sequenceand expressed. In some embodiments, the enzyme is active. In someembodiments, a murine 1L3 DNase-Ig-RNase hybrid nuclease is constructedand expressed. In some embodiments, the molecule includes human 1L3-Ig,human 1L3-Ig-RNase, and/or human RNase-Ig-1L3.

In some embodiments, the hybrid nuclease molecule includes DNase1-Ig. Insome embodiments, a naturally occurring variant allele, A114F, whichshows reduced sensitivity to actin is included in a DNase1-Ig hybridnuclease molecule. In some embodiments, this mutation is introduced intoa hybrid nuclease molecule to generate a more stable derivative of humanDNase1. In some embodiments, a DNase1-linker-Ig containing a 20 or 25 aalinker domain is made. In some embodiments, hybrid nuclease moleculesinclude RNase-Ig-linker-DNase1 where the DNase1 domain is located at theCOOH side of the Ig Fc domain. In some embodiments, hybrid nucleasemolecules are made that incorporate DNase1 and include:DNase1-linker-Ig-linker2-RNase, and/or RNase-Ig-linker-DNase1.

Another aspect of the present invention is to use gene therapy methodsfor treating or preventing disorders, diseases, and conditions with oneor more hybrid nuclease molecules. The gene therapy methods relate tothe introduction of hybrid nuclease molecule nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofthe polypeptide or polypeptides of the present invention. This methodcan include introduction of one or more polynucleotides encoding ahybrid nuclease molecule polypeptide of the present inventionoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.

In gene therapy applications, hybrid nuclease molecule genes areintroduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product. “Gene therapy” includes bothconventional gene therapies where a lasting effect is achieved by asingle treatment, and the administration of gene therapeutic agents,which involves the one time or repeated administration of atherapeutically effective DNA or mRNA. The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

Fc Domains

In some embodiments, a hybrid nuclease molecule includes an Fc domain.The Fc domain does not contain a variable region that binds to antigen.In embodiments, the Fc domain does not contain a variable region. Fcdomains useful for producing the hybrid nuclease molecules of thepresent invention may be obtained from a number of different sources. Inpreferred embodiments, an Fc domain of the hybrid nuclease molecule isderived from a human immunoglobulin. It is understood, however, that theFc domain may be derived from an immunoglobulin of another mammalianspecies, including for example, a rodent (e.g. a mouse, rat, rabbit,guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.Moreover, the hybrid nuclease molecule Fc domain or portion thereof maybe derived from any immunoglobulin class, including IgM, IgG, IgD, IgA,and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, andIgG4. In a preferred embodiment, the human isotype IgG1 is used.

In some aspects, a hybrid nuclease molecule includes a mutant Fc domain.In some aspects, a hybrid nuclease molecule includes a mutant, IgG1 Fcdomain In some aspects, a mutant Fc domain comprises one or moremutations in the hinge, CH2, and/or CH3 domains. In some aspects, amutant Fc domain includes a P238S mutation. In some aspects, a mutant Fcdomain includes a P331S mutation. In some aspects, a mutant Fc domainincludes a P238S mutation and a P331S mutation. In some aspects, amutant Fc domain comprises P238S and/or P331S, and may include mutationsin one or more of the three hinge cysteines. In some aspects, a mutantFc domain comprises P238S and/or P331S, and/or one or more mutations inthe three hinge cysteines. In some aspects, a mutant Fc domain comprisesP238S and/or P331S, and/or mutations in a hinge cysteine to SCC or inthe three hinge cysteines to SSS. In some aspects, a mutant Fc domaincomprises P238S and P331S and mutations in at least one of the threehinge cysteines. In some aspects, a mutant Fc domain comprises P238S andP331S and SCC. In some aspects, a mutant Fc domain comprises P238S andP331S and SSS. In some aspects, a mutant Fc domain includes P238S andSCC or SSS. In some aspects, a mutant Fc domain includes P331S and SCCor SSS. In some aspects, a mutant Fc domain includes mutations in one ormore of the three hinge cysteines. In some aspects, a mutant Fc domainincludes mutations in the three hinge cysteines. In some aspects, amutant Fc domain includes mutations in one of the three hinge cysteinesto SCC. In some aspects, a mutant Fc domain includes SCC. In someaspects, a mutant Fc domain includes mutations in the three hingecysteines to SSS. In some aspects, a mutant Fc domain includes SSS. Insome aspects, a nucleic acid sequence encoding a mutant Fc domain isshown in SEQ ID NOs 59, 71, 73, 75, 87, or 89. In some aspects, a mutantFc domain is as shown as in SEQ ID NOs 60, 72, 74, 76, 88, or 90. Insome aspects, a nucleic acid sequence encoding a hybrid nucleasemolecule is as shown in SEQ ID NOs 61, 63, 65, 67, 69, 77, 79, 81, 83,85, 91, 93, 95 or 97. In some aspects, a hybrid nuclease molecule is asshown in SEQ ID NOs 62, 64, 66, 68, 70, 78, 80, 82, 84,86, 92, 94, 96,or 98.

A variety of Fc domain gene sequences (e.g., human constant region genesequences) are available in the form of publicly accessible deposits.Constant region domains comprising an Fc domain sequence can be selectedhaving a particular effector function (or lacking a particular effectorfunction) or with a particular modification to reduce immunogenicity.Many sequences of antibodies and antibody-encoding genes have beenpublished and suitable Fc domain sequences (e.g. hinge, CH2, and/or CH3sequences, or portions thereof) can be derived from these sequencesusing art recognized techniques. The genetic material obtained using anyof the foregoing methods may then be altered or synthesized to obtainpolypeptides of the present invention. It will further be appreciatedthat the scope of this invention encompasses alleles, variants andmutations of constant region DNA sequences.

Fc domain sequences can be cloned, e.g., using the polymerase chainreaction and primers which are selected to amplify the domain ofinterest. To clone an Fc domain sequence from an antibody, mRNA can beisolated from hybridoma, spleen, or lymph cells, reverse transcribedinto DNA, and antibody genes amplified by PCR. PCR amplification methodsare described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; 4,965,188; and in, e.g., “PCR Protocols: A Guide to Methodsand Applications” Innis et al. eds., Academic Press, San Diego, Calif.(1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol.217:270). PCR may be initiated by consensus constant region primers orby more specific primers based on the published heavy and light chainDNA and amino acid sequences. As discussed above, PCR also may be usedto isolate DNA clones encoding the antibody light and heavy chains. Inthis case the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes. Numerous primersets suitable for amplification of antibody genes are known in the art(e.g., 5′ primers based on the N-terminal sequence of purifiedantibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapidamplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods173:33); antibody leader sequences (Larrick et al. 1989 Biochem.Biophys. Res. Commun. 160:1250). The cloning of antibody sequences isfurther described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan.25, 1995, which is incorporated by reference herein.

The hybrid nuclease molecules of the invention may comprise one or moreFc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc domains). Inone embodiment, the Fc domains may be of different types. In oneembodiment, at least one Fc domain present in the hybrid nucleasemolecule comprises a hinge domain or portion thereof. In anotherembodiment, the hybrid nuclease molecule of the invention comprises atleast one Fc domain which comprises at least one CH2 domain or portionthereof. In another embodiment, the hybrid nuclease molecule of theinvention comprises at least one Fc domain which comprises at least oneCH3 domain or portion thereof. In another embodiment, the hybridnuclease molecule of the invention comprises at least one Fc domainwhich comprises at least one CH4 domain or portion thereof. In anotherembodiment, the hybrid nuclease molecule of the invention comprises atleast one Fc domain which comprises at least one hinge domain or portionthereof and at least one CH2 domain or portion thereof (e.g, in thehinge-CH2 orientation). In another embodiment, the hybrid nucleasemolecule of the invention comprises at least one Fc domain whichcomprises at least one CH2 domain or portion thereof and at least oneCH3 domain or portion thereof (e.g, in the CH2-CH3 orientation). Inanother embodiment, the hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising at least one hinge domain orportion thereof, at least one CH2 domain or portion thereof, and leastone CH3 domain or portion thereof, for example in the orientationhinge-CH2-CH3, hinge-CH3-CH2, or CH2-CH3-hinge.

In certain embodiments, the hybrid nuclease molecule comprises at leastone complete Fc region derived from one or more immunoglobulin heavychains (e.g., an Fc domain including hinge, CH2, and CH3 domains,although these need not be derived from the same antibody). In otherembodiments, the hybrid nuclease molecule comprises at least twocomplete Fc domains derived from one or more immunoglobulin heavychains. In preferred embodiments, the complete Fc domain is derived froma human IgG immunoglobulin heavy chain (e.g., human IgG1).

In another embodiment, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising a complete CH3 domain. Inanother embodiment, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising a complete CH2 domain. Inanother embodiment, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising at least a CH3 domain, andat least one of a hinge region, and a CH2 domain. In one embodiment, ahybrid nuclease molecule of the invention comprises at least one Fcdomain comprising a hinge and a CH3 domain. In another embodiment, ahybrid nuclease molecule of the invention comprises at least one Fcdomain comprising a hinge, a CH2, and a CH3 domain. In preferredembodiments, the Fc domain is derived from a human IgG immunoglobulinheavy chain (e.g., human IgG1).

The constant region domains or portions thereof making up an Fc domainof a hybrid nuclease molecule of the invention may be derived fromdifferent immunoglobulin molecules. For example, a polypeptide of theinvention may comprise a CH2 domain or portion thereof derived from anIgG1 molecule and a CH3 region or portion thereof derived from an IgG3molecule. In another example, a hybrid nuclease molecule can comprise anFc domain comprising a hinge domain derived, in part, from an IgG1molecule and, in part, from an IgG3 molecule. As set forth herein, itwill be understood by one of ordinary skill in the art that an Fc domainmay be altered such that it varies in amino acid sequence from anaturally occurring antibody molecule.

In another embodiment, a hybrid nuclease molecule of the inventioncomprises one or more truncated Fc domains that are nonethelesssufficient to confer Fc receptor (FcR) binding properties to the Fcregion. Thus, an Fc domain of a hybrid nuclease molecule of theinvention may comprise or consist of an FcRn binding portion. FcRnbinding portions may be derived from heavy chains of any isotype,including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRn bindingportion from an antibody of the human isotype IgG1 is used. In anotherembodiment, an FcRn binding portion from an antibody of the humanisotype IgG4 is used.

In one embodiment, a hybrid nuclease molecule of the invention lacks oneor more constant region domains of a complete Fc region, i.e., they arepartially or entirely deleted. In a certain embodiments hybrid nucleasemolecules of the invention will lack an entire CH2 domain (ΔCH2constructs). Those skilled in the art will appreciate that suchconstructs may be preferred due to the regulatory properties of the CH2domain on the catabolic rate of the antibody. In certain embodiments,hybrid nuclease molecules of the invention comprise CH2 domain-deletedFc regions derived from a vector (e.g., from IDEC Pharmaceuticals, SanDiego) encoding an IgG1 human constant region domain (see, e.g., WO02/060955A2 and WO02/096948A2). This exemplary vector is engineered todelete the CH2 domain and provide a synthetic vector expressing adomain-deleted IgG1 constant region. It will be noted that theseexemplary constructs are preferably engineered to fuse a binding CH3domain directly to a hinge region of the respective Fc domain.

In other constructs it may be desirable to provide a peptide spacerbetween one or more constituent Fc domains. For example, a peptidespacer may be placed between a hinge region and a CH2 domain and/orbetween a CH2 and a CH3 domain. For example, compatible constructs couldbe expressed wherein the CH2 domain has been deleted and the remainingCH3 domain (synthetic or unsynthetic) is joined to the hinge region witha 1-20, 1-10, or 1-5 amino acid peptide spacer. Such a peptide spacermay be added, for instance, to ensure that the regulatory elements ofthe constant region domain remain free and accessible or that the hingeregion remains flexible. Preferably, any linker peptide compatible withthe instant invention will be relatively non-immunogenic and not preventproper folding of the Fc.

Changes to Fc Amino Acids

In certain embodiments, an Fc domain employed in a hybrid nucleasemolecule of the invention is altered or modified, e.g., by amino acidmutation (e.g., addition, deletion, or substitution). As used herein,the term “Fc domain variant” refers to an Fc domain having at least oneamino acid modification, such as an amino acid substitution, as comparedto the wild-type Fc from which the Fc domain is derived. For example,wherein the Fc domain is derived from a human IgG1 antibody, a variantcomprises at least one amino acid mutation (e.g., substitution) ascompared to a wild type amino acid at the corresponding position of thehuman IgG1 Fc region.

The amino acid substitution(s) of an Fc variant may be located at aposition within the Fc domain referred to as corresponding to theportion number that that residue would be given in an Fc region in anantibody.

In one embodiment, the Fc variant comprises a substitution at an aminoacid position located in a hinge domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

In certain embodiments, the hybrid nuclease molecules of the inventioncomprise an Fc variant comprising more than one amino acid substitution.The hybrid nuclease molecules of the invention may comprise, forexample, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.Preferably, the amino acid substitutions are spatially positioned fromeach other by an interval of at least 1 amino acid position or more, forexample, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions ormore. More preferably, the engineered amino acids are spatiallypositioned apart from each other by an interval of at least 5, 10, 15,20, or 25 amino acid positions or more.

In certain embodiments, the Fc variant confers an improvement in atleast one effector function imparted by an Fc domain comprising saidwild-type Fc domain (e.g., an improvement in the ability of the Fcdomain to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) orcomplement proteins (e.g. C1q), or to trigger antibody-dependentcytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity(CDCC)). In other embodiments, the Fc variant provides an engineeredcysteine residue.

In some aspects, an Fc domain includes changes in the region betweenamino acids 234-238, including the sequence LLGGP at the beginning ofthe CH2 domain. In some aspects, an Fc variant alters Fc mediatedeffector function, particularly ADCC, and/or decrease binding avidityfor Fc receptors. In some aspects, sequence changes closer to theCH2-CH3 junction, at positions such as K322 or P331 can eliminatecomplement mediated cytotoxicity and/or alter avidity for FcR binding.In some aspects, an Fc domain incorporates changes at residues P238 andP331, e.g., changing the wild type prolines at these positions toserine. In some aspects, alterations in the hinge region at one or moreof the three hinge cysteines, to encode CCC, SCC, SSC, SCS, or SSS atthese residues can also affect FcR binding and molecular homogeneity,e.g., by elimination of unpaired cysteines that may destabilize thefolded protein.

The hybrid nuclease molecules of the invention may employ art-recognizedFc variants which are known to impart an improvement in effectorfunction and/or FcR binding. Specifically, a hybrid nuclease molecule ofthe invention may include, for example, a change (e.g., a substitution)at one or more of the amino acid positions disclosed in InternationalPCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1,WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2,WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351 A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S.Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871;6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124;6,737,056; 6,821,505; 6,998,253; 7,083,784; and 7,317,091, each of whichis incorporated by reference herein. In one embodiment, the specificchange (e.g., the specific substitution of one or more amino acidsdisclosed in the art) may be made at one or more of the disclosed aminoacid positions. In another embodiment, a different change at one or moreof the disclosed amino acid positions (e.g., the different substitutionof one or more amino acid position disclosed in the art) may be made.

Other amino acid mutations in the Fc domain are contemplated to reducebinding to the Fc gamma receptor and Fc gamma receptor subtypes. Forexample, mutations at positions 238, 239, 248, 249, 252, 254, 255, 256,258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290,292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 322, 324,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373, 376,378, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region can alter binding as described in U.S. Pat. No.6,737,056, issued May 18, 2004, incorporated herein by reference in itsentirety. This patent reported that changing Pro331 in IgG3 to Serresulted in six fold lower affinity as compared to unmutated IgG3,indicating the involvement of Pro331 in Fc gamma RI binding. Inaddition, amino acid modifications at positions 234, 235, 236, and 237,297, 318, 320 and 322 are disclosed as potentially altering receptorbinding affinity in U.S. Pat. No. 5,624,821, issued Apr. 29, 1997 andincorporated herein by reference in its entirety.

Further mutations contemplated for use include, e.g., those described inU.S. Pat. App. Pub. No. 2006/0235208, published Oct. 19, 2006 andincorporated herein by reference in its entirety. This publicationsdescribe Fc variants that exhibit reduced binding to Fc gamma receptors,reduced antibody dependent cell-mediated cytotoxicity, or reducedcomplement dependent cytotoxicity, that comprise at least one amino acidmodification in the Fc region, including 232G, 234G, 234H, 235D, 235G,235H, 236I, 236N, 236P, 236R, 237K, 237L, 237N, 237P, 238K, 239R, 265G,267R, 269R, 270H, 297S, 299A, 299I, 299V, 325A, 325L, 327R, 328R, 329K,330I, 330L, 330N, 330P, 330R, and 331L (numbering is according to the EUindex), as well as double mutants 236R/237K, 236R/325L, 236R/328R,237K/325L, 237K/328R, 325L/328R, 235G/236R, 267R/269R, 234G/235G,236R/237K/325L, 236R/325L/328R, 235G/236R/237K, and 237K/325L/328R.Other mutations contemplated for use as described in this publicationinclude 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 235I, 235S, 236S,239D, 246H, 255Y, 258H, 260H, 264I, 267D, 267E, 268D, 268E, 272H, 272I,272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I, 327D, 327A,328A, 328D, 328E, 328F, 328I, 328M, 328N, 328Q, 328T, 328V, 328Y, 330I,330L, 330Y, 332D, 332E, 335D, an insertion of G between positions 235and 236, an insertion of A between positions 235 and 236, an insertionof S between positions 235 and 236, an insertion of T between positions235 and 236, an insertion of N between positions 235 and 236, aninsertion of D between positions 235 and 236, an insertion of V betweenpositions 235 and 236, an insertion of L between positions 235 and 236,an insertion of G between positions 235 and 236, an insertion of Abetween positions 235 and 236, an insertion of S between positions 235and 236, an insertion of T between positions 235 and 236, an insertionof N between positions 235 and 236, an insertion of D between positions235 and 236, an insertion of V between positions 235 and 236, aninsertion of L between positions 235 and 236, an insertion of G betweenpositions 297 and 298, an insertion of A between positions 297 and 298,an insertion of S between positions 297 and 298, an insertion of Dbetween positions 297 and 298, an insertion of G between positions 326and 327, an insertion of A between positions 326 and 327, an insertionof T between positions 326 and 327, an insertion of D between positions326 and 327, and an insertion of E between positions 326 and 327(numbering is according to the EU index). Additionally, mutationsdescribed in U.S. Pat. App. Pub. No. 2006/0235208 include 227G/332E,234D/332E, 234E/332E, 234Y/332E, 234I/332E, 234G/332E, 235I/332E,235S/332E, 235D/332E, 235E/332E, 236S/332E, 236A/332E, 236S/332D,236A/332D, 239D/268E, 246H/332E, 255Y/332E, 258H/332E, 260H/332E,264I/332E, 267E/332E, 267D/332E, 268D/332D, 268E/332D, 268E/332E,268D/332E, 268E/330Y, 268D/330Y, 272R/332E, 272H/332E, 283H/332E,284E/332E, 293R/332E, 295E/332E, 304T/332E, 324I/332E, 324G/332E,324I/332D, 324G/332D, 327D/332E, 328A/332E, 328T/332E, 328V/332E,328I/332E, 328F/332E, 328Y/332E, 328M/332E, 328D/332E, 328E/332E,328N/332E, 328Q/332E, 328A/332D, 328T/332D, 328V/332D, 328I/332D,328F/332D, 328Y/332D, 328M/332D, 328D/332D, 328E/332D, 328N/332D,328Q/332D, 330L/332E, 330Y/332E, 330I/332E, 332D/330Y, 335D/332E,239D/332E, 239D/332E/330Y, 239D/332E/330L, 239D/332E/330I,239D/332E/268E, 239D/332E/268D, 239D/332E/327D, 239D/332E/284E,239D/268E/330Y, 239D/332E/268E/330Y, 239D/332E/327A,239D/332E/268E/327A, 239D/332E/330Y/327A, 332E/330Y/268 E/327A,239D/332E/268E/330Y/327A, Insert G>297-298/332E, Insert A>297-298/332E,Insert S>297-298/332E, Insert D>297-298/332E, Insert G>326-327/332E,Insert A>326-327/332E, Insert T>326-327/332E, Insert D>326-327/332E,Insert E>326-327/332E, Insert G>235-236/332E, Insert A>235-236/332E,Insert S>235-236/332E, Insert T>235-236/332E, Insert N>235-236/332E,Insert D>235-236/332E, Insert V>235-236/332E, Insert L>235-236/332E,Insert G>235-236/332D, Insert A>235-236/332D, Insert S>235-236/332D,Insert T>235-236/332D, Insert N>235-236/332D, Insert D>235-236/332D,Insert V>235-236/332D, and Insert L>235-236/332D (numbering according tothe EU index) are contemplated for use. The mutant L234A/L235A isdescribed, e.g., in U.S. Pat. App. Pub. No. 2003/0108548, published Jun.12, 2003 and incorporated herein by reference in its entirety. Inembodiments, the described modifications are included eitherindividually or in combination.

In certain embodiments, a hybrid nuclease molecule of the inventioncomprises an amino acid substitution to an Fc domain which alters theantigen-independent effector functions of the antibody, in particularthe circulating half-life of the antibody. Such hybrid nucleasemolecules exhibit either increased or decreased binding to FcRn whencompared to hybrid nuclease molecules lacking these substitutions and,therefore, have an increased or decreased half-life in serum,respectively. Fc variants with improved affinity for FcRn areanticipated to have longer serum half-lives, and such molecules haveuseful applications in methods of treating mammals where long half-lifeof the administered polypeptide is desired, e.g., to treat a chronicdisease or disorder. In contrast, Fc variants with decreased FcRnbinding affinity are expected to have shorter half-lives, and suchmolecules are also useful, for example, for administration to a mammalwhere a shortened circulation time may be advantageous, e.g. for in vivodiagnostic imaging or in situations where the starting polypeptide hastoxic side effects when present in the circulation for prolongedperiods. Fc variants with decreased FcRn binding affinity are also lesslikely to cross the placenta and, thus, are also useful in the treatmentof diseases or disorders in pregnant women. In addition, otherapplications in which reduced FcRn binding affinity may be desiredinclude those applications in which localization the brain, kidney,and/or liver is desired. In one exemplary embodiment, the hybridnuclease molecules of the invention exhibit reduced transport across theepithelium of kidney glomeruli from the vasculature. In anotherembodiment, the hybrid nuclease molecules of the invention exhibitreduced transport across the blood brain barrier (BBB) from the brain,into the vascular space. In one embodiment, a hybrid nuclease moleculewith altered FcRn binding comprises at least one Fc domain (e.g., one ortwo Fc domains) having one or more amino acid substitutions within the“FcRn binding loop” of an Fc domain. Exemplary amino acid substitutionswhich altered FcRn binding activity are disclosed in International PCTPublication No. WO05/047327 which is incorporated by reference herein.

In other embodiments, a hybrid nuclease molecule of the inventioncomprises an Fc variant comprising an amino acid substitution whichalters the antigen-dependent effector functions of the polypeptide, inparticular ADCC or complement activation, e.g., as compared to a wildtype Fc region. In exemplary embodiment, said hybrid nuclease moleculesexhibit altered binding to an Fc gamma receptor (e.g., CD16). Suchhybrid nuclease molecules exhibit either increased or decreased bindingto FcR gamma when compared to wild-type polypeptides and, therefore,mediate enhanced or reduced effector function, respectively. Fc variantswith improved affinity for FcγRs are anticipated to enhance effectorfunction, and such molecules have useful applications in methods oftreating mammals where target molecule destruction is desired. Incontrast, Fc variants with decreased FcγR binding affinity are expectedto reduce effector function, and such molecules are also useful, forexample, for treatment of conditions in which target cell destruction isundesirable, e.g., where normal cells may express target molecules, orwhere chronic administration of the polypeptide might result in unwantedimmune system activation. In one embodiment, the polypeptide comprisingan Fc exhibits at least one altered antigen-dependent effector functionselected from the group consisting of opsonization, phagocytosis,complement dependent cytotoxicity, antigen-dependent cellularcytotoxicity (ADCC), or effector cell modulation as compared to apolypeptide comprising a wild type Fc region.

In one embodiment the hybrid nuclease molecule exhibits altered bindingto an activating FcγR (e.g. FcγI, FcγIIa, or FcγRIIIa). In anotherembodiment, the hybrid nuclease molecule exhibits altered bindingaffinity to an inhibitory FcγR (e.g. FcγRIIb). Exemplary amino acidsubstitutions which altered FcR or complement binding activity aredisclosed in International PCT Publication No. WO05/063815 which isincorporated by reference herein.

A hybrid nuclease molecule of the invention may also comprise an aminoacid substitution which alters the glycosylation of the hybrid nucleasemolecule. For example, the Fc domain of the hybrid nuclease molecule maycomprise an Fc domain having a mutation leading to reduced glycosylation(e.g., N- or O-linked glycosylation) or may comprise an alteredglycoform of the wild-type Fc domain (e.g., a low fucose or fucose-freeglycan). In another embodiment, the hybrid nuclease molecule has anamino acid substitution near or within a glycosylation motif, forexample, an N-linked glycosylation motif that contains the amino acidsequence NXT or NXS. Exemplary amino acid substitutions which reduce oralter glycosylation are disclosed in International PCT Publication No.WO05/018572 and US Patent Publication No. 2007/0111281, which areincorporated by reference herein.

In other embodiments, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain having engineered cysteine residue oranalog thereof which is located at the solvent-exposed surface.Preferably the engineered cysteine residue or analog thereof does notinterfere with an effector function conferred by the Fc. Morepreferably, the alteration does not interfere with the ability of the Fcto bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) or complementproteins (e.g. C1q), or to trigger immune effector function (e.g.,antibody-dependent cytotoxicity (ADCC), phagocytosis, orcomplement-dependent cytotoxicity (CDCC)). In preferred embodiments, thehybrid nuclease molecules of the invention comprise an Fc domaincomprising at least one engineered free cysteine residue or analogthereof that is substantially free of disulfide bonding with a secondcysteine residue. Any of the above engineered cysteine residues oranalogs thereof may subsequently be conjugated to a functional domainusing art-recognized techniques (e.g., conjugated with a thiol-reactiveheterobifunctional linker).

In one embodiment, the hybrid nuclease molecule of the invention maycomprise a genetically fused Fc domain having two or more of itsconstituent Fc domains independently selected from the Fc domainsdescribed herein. In one embodiment, the Fc domains are the same. Inanother embodiment, at least two of the Fc domains are different. Forexample, the Fc domains of the hybrid nuclease molecules of theinvention comprise the same number of amino acid residues or they maydiffer in length by one or more amino acid residues (e.g., by about 5amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about10 residues, about 15 residues, about 20 residues, about 30 residues,about 40 residues, or about 50 residues). In yet other embodiments, theFc domains of the hybrid nuclease molecules of the invention may differin sequence at one or more amino acid positions. For example, at leasttwo of the Fc domains may differ at about 5 amino acid positions (e.g.,1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15positions, about 20 positions, about 30 positions, about 40 positions,or about 50 positions).

Linker Domains

In some embodiments, a hybrid nuclease molecule includes a linkerdomain. In some embodiments, a hybrid nuclease molecule includes aplurality of linker domains. In some embodiments, the linker domain is apolypeptide linker. In certain aspects, it is desirable to employ apolypeptide linker to fuse one or more Fc domains to one or morenuclease domains to form a hybrid nuclease molecule.

In one embodiment, the polypeptide linker is synthetic. As used hereinthe term “synthetic” with respect to a polypeptide linker includespeptides (or polypeptides) which comprise an amino acid sequence (whichmay or may not be naturally occurring) that is linked in a linearsequence of amino acids to a sequence (which may or may not be naturallyoccurring) (e.g., an Fc domain sequence) to which it is not naturallylinked in nature. For example, the polypeptide linker may comprisenon-naturally occurring polypeptides which are modified forms ofnaturally occurring polypeptides (e.g., comprising a mutation such as anaddition, substitution or deletion) or which comprise a first amino acidsequence (which may or may not be naturally occurring). The polypeptidelinkers of the invention may be employed, for instance, to ensure thatFc domains are juxtaposed to ensure proper folding and formation of afunctional Fc domain. Preferably, a polypeptide linker compatible withthe instant invention will be relatively non-immunogenic and not inhibitany non-covalent association among monomer subunits of a bindingprotein.

In certain embodiments, the hybrid nuclease molecules of the inventionemploy a polypeptide linker to join any two or more domains in frame ina single polypeptide chain. In one embodiment, the two or more domainsmay be independently selected from any of the Fc domains or nucleasedomains discussed herein. For example, in certain embodiments, apolypeptide linker can be used to fuse identical Fc domains, therebyforming a homomeric Fc region. In other embodiments, a polypeptidelinker can be used to fuse different Fc domains (e.g. a wild-type Fcdomain and a Fc domain variant), thereby forming a heteromeric Fcregion. In other embodiments, a polypeptide linker of the invention canbe used to genetically fuse the C-terminus of a first Fc domain (e.g. ahinge domain or portion thereof, a CH2 domain or portion thereof, acomplete CH3 domain or portion thereof, a FcRn binding portion, an FcγRbinding portion, a complement binding portion, or portion thereof) tothe N-terminus of a second Fc domain (e.g., a complete Fc domain).

In one embodiment, a polypeptide linker comprises a portion of an Fcdomain. For example, in one embodiment, a polypeptide linker cancomprise an immunoglobulin hinge domain of an IgG1, IgG2, IgG3, and/orIgG4 antibody. In another embodiment, a polypeptide linker can comprisea CH2 domain of an IgG1, IgG2, IgG3, and/or IgG4 antibody. In otherembodiments, a polypeptide linker can comprise a CH3 domain of an IgG1,IgG2, IgG3, and/or IgG4 antibody. Other portions of an immunoglobulin(e.g. a human immunoglobulin) can be used as well. For example, apolypeptide linker can comprise a CH1 domain or portion thereof, a CLdomain or portion thereof, a VH domain or portion thereof, or a VLdomain or portion thereof. Said portions can be derived from anyimmunoglobulin, including, for example, an IgG1, IgG2, IgG3, and/or IgG4antibody.

In exemplary embodiments, a polypeptide linker can comprise at least aportion of an immunoglobulin hinge region. In one embodiment, apolypeptide linker comprises an upper hinge domain (e.g., an IgG1, anIgG2, an IgG3, or IgG4 upper hinge domain) In another embodiment, apolypeptide linker comprises a middle hinge domain (e.g., an IgG1, anIgG2, an IgG3, or an IgG4 middle hinge domain). In another embodiment, apolypeptide linker comprises a lower hinge domain (e.g., an IgG1, anIgG2, an IgG3, or an IgG4 lower hinge domain).

In other embodiments, polypeptide linkers can be constructed whichcombine hinge elements derived from the same or different antibodyisotypes. In one embodiment, the polypeptide linker comprises a chimerichinge comprising at least a portion of an IgG1 hinge region and at leasta portion of an IgG2 hinge region. In one embodiment, the polypeptidelinker comprises a chimeric hinge comprising at least a portion of anIgG1 hinge region and at least a portion of an IgG3 hinge region. Inanother embodiment, a polypeptide linker comprises a chimeric hingecomprising at least a portion of an IgG1 hinge region and at least aportion of an IgG4 hinge region. In one embodiment, the polypeptidelinker comprises a chimeric hinge comprising at least a portion of anIgG2 hinge region and at least a portion of an IgG3 hinge region. In oneembodiment, the polypeptide linker comprises a chimeric hinge comprisingat least a portion of an IgG2 hinge region and at least a portion of anIgG4 hinge region. In one embodiment, the polypeptide linker comprises achimeric hinge comprising at least a portion of an IgG1 hinge region, atleast a portion of an IgG2 hinge region, and at least a portion of anIgG4 hinge region. In another embodiment, a polypeptide linker cancomprise an IgG1 upper and middle hinge and a single IgG3 middle hingerepeat motif. In another embodiment, a polypeptide linker can comprisean IgG4 upper hinge, an IgG1 middle hinge and a IgG2 lower hinge.

In another embodiment, a polypeptide linker comprises or consists of agly-ser linker. As used herein, the term “gly-ser linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser linker comprises an amino acid sequence of the formula(Gly₄Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). Apreferred gly/ser linker is (Gly₄Ser)4. Another preferred gly/ser linkeris (Gly₄Ser)3. Another preferred gly/ser linker is (Gly₄Ser)5. Incertain embodiments, the gly-ser linker may be inserted between twoother sequences of the polypeptide linker (e.g., any of the polypeptidelinker sequences described herein). In other embodiments, a gly-serlinker is attached at one or both ends of another sequence of thepolypeptide linker (e.g., any of the polypeptide linker sequencesdescribed herein). In yet other embodiments, two or more gly-ser linkerare incorporated in series in a polypeptide linker. In one embodiment, apolypeptide linker of the invention comprises at least a portion of anupper hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4molecule), at least a portion of a middle hinge region (e.g., derivedfrom an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of gly/seramino acid residues (e.g., a gly/ser linker such as (Gly₄Ser)n).

In one embodiment, a polypeptide linker of the invention comprises anon-naturally occurring immunoglobulin hinge region domain, e.g., ahinge region domain that is not naturally found in the polypeptidecomprising the hinge region domain and/or a hinge region domain that hasbeen altered so that it differs in amino acid sequence from a naturallyoccurring immunoglobulin hinge region domain. In one embodiment,mutations can be made to hinge region domains to make a polypeptidelinker of the invention. In one embodiment, a polypeptide linker of theinvention comprises a hinge domain which does not comprise a naturallyoccurring number of cysteines, i.e., the polypeptide linker compriseseither fewer cysteines or a greater number of cysteines than a naturallyoccurring hinge molecule.

In other embodiments, a polypeptide linker of the invention comprises abiologically relevant peptide sequence or a sequence portion thereof.For example, a biologically relevant peptide sequence may include, butis not limited to, sequences derived from an anti-rejection oranti-inflammatory peptide. Said anti-rejection or anti-inflammatorypeptides may be selected from the group consisting of a cytokineinhibitory peptide, a cell adhesion inhibitory peptide, a thrombininhibitory peptide, and a platelet inhibitory peptide. In a onepreferred embodiment, a polypeptide linker comprises a peptide sequenceselected from the group consisting of an IL-1 inhibitory or antagonistpeptide sequence, an erythropoietin (EPO)-mimetic peptide sequence, athrombopoietin (TPO)-mimetic peptide sequence, G-CSF mimetic peptidesequence, a TNF-antagonist peptide sequence, an integrin-binding peptidesequence, a selectin antagonist peptide sequence, an anti-pathogenicpeptide sequence, a vasoactive intestinal peptide (VIP) mimetic peptidesequence, a calmodulin antagonist peptide sequence, a mast cellantagonist, a SH3 antagonist peptide sequence, an urokinase receptor(UKR) antagonist peptide sequence, a somatostatin or cortistatin mimeticpeptide sequence, and a macrophage and/or T-cell inhibiting peptidesequence. Exemplary peptide sequences, any one of which may be employedas a polypeptide linker, are disclosed in U.S. Pat. No. 6,660,843, whichis incorporated by reference herein.

It will be understood that variant forms of these exemplary polypeptidelinkers can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequenceencoding a polypeptide linker such that one or more amino acidsubstitutions, additions or deletions are introduced into thepolypeptide linker. For example, mutations may be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Polypeptide linkers of the invention are at least one amino acid inlength and can be of varying lengths. In one embodiment, a polypeptidelinker of the invention is from about 1 to about 50 amino acids inlength. As used in this context, the term “about” indicates +/−two aminoacid residues. Since linker length must be a positive integer, thelength of from about 1 to about 50 amino acids in length, means a lengthof from 1 to 48-52 amino acids in length. In another embodiment, apolypeptide linker of the invention is from about 10-20 amino acids inlength. In another embodiment, a polypeptide linker of the invention isfrom about 15 to about 50 amino acids in length.

In another embodiment, a polypeptide linker of the invention is fromabout 20 to about 45 amino acids in length. In another embodiment, apolypeptide linker of the invention is from about 15 to about 25 aminoacids in length. In another embodiment, a polypeptide linker of theinvention is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56 ,57, 58, 59, 60, or more amino acids in length.

Polypeptide linkers can be introduced into polypeptide sequences usingtechniques known in the art. Modifications can be confirmed by DNAsequence analysis. Plasmid DNA can be used to transform host cells forstable production of the polypeptides produced.

Nuclease Domains

In certain aspects, a hybrid nuclease molecule includes a nucleasedomain. Accordingly, the hybrid nuclease molecules of the inventiontypically comprise at least one nuclease domain and at least one linkedFc domain. In certain aspects, a hybrid nuclease molecule includes aplurality of nuclease domains.

In some embodiments, a nuclease domain is substantially all or at leastan enzymatically active fragment of a DNase. In some embodiments, theDNase is a Type I secreted DNase, preferably a human DNase such asDNase 1. Exemplary DNase 1 domains are set forth in SEQ ID NOs 48-53 and102. An exemplary human DNase 1 is described at UniProtKB entry P24855(SEQ ID NO:49 and 102). In some embodiments, the DNase is DNase 1 and/ora DNase 1-like (DNaseL) enzyme, 1-3. An exemplary human Dnase 1-likeenzyme, 1-3 is described at UniProtKB entry Q13609 (SEQ ID NO:57 and103). In some embodiments, the DNase is TREX1 (Three prime repairexonuclease 1). An exemplary human TREX1 is described at UniProtKB entryQ9NSU2 (SEQ ID NO:104). Preferably the human TREX1 is a C-terminaltruncated human TREX1 lacking intracellular nuclear targeting sequences,e.g., a human TREX1 lacking 72 C-terminal amino acids as set forth inSEQ ID NO:105.

In some embodiments, a nuclease domain is substantially all or at leastan enzymatically active fragment of an RNase. In some embodiments, theRNase is an extracellular or secretory RNase of the RNase A superfamily,e.g., RNase A, preferably a human pancreatic RNase. An exemplary humanRnase is described at UniProtKB entry P07998 (SEQ ID NO:58 and 101).

In one embodiment, the nuclease domain is operably linked (e.g.,chemically conjugated or genetically fused (e.g., either directly or viaa polypeptide linker)) to the N-terminus of an Fc domain. In anotherembodiment, the nuclease domain is operably linked (e.g., chemicallyconjugated or genetically fused (e.g., either directly or via apolypeptide linker)) to the C-terminus of an Fc domain. In otherembodiments, a nuclease domain is operably linked (e.g., chemicallyconjugated or genetically fused (e.g., either directly or via apolypeptide linker)) via an amino acid side chain of an Fc domain. Incertain exemplary embodiments, the nuclease domain is fused to an Fcdomain via a human immunoglobulin hinge domain or portion thereof.

In certain embodiments, the hybrid nuclease molecules of the inventioncomprise two or more nuclease domains and at least one Fc domain. Forexample, nuclease domains may be operably linked to both the N-terminusand C-terminus of an Fc domain. In other exemplary embodiments, nucleasedomains may be operably linked to both the N- and C-terminal ends ofmultiple Fc domains (e.g., two, three, four, five, or more Fc domains)which are linked together in series to form a tandem array of Fcdomains.

In other embodiments, two or more nuclease domains are linked to eachother (e.g., via a polypeptide linker) in series, and the tandem arrayof nuclease domains is operably linked (e.g., chemically conjugated orgenetically fused (e.g., either directly or via a polypeptide linker))to either the C-terminus or the N-terminus of a Fc domain or a tandemarray of Fc domains. In other embodiments, the tandem array of nucleasedomains is operably linked to both the C-terminus and the N-terminus ofa Fc domain or a tandem array of Fc domains.

In other embodiments, one or more nuclease domains may be insertedbetween two Fc domains. For example, one or more nuclease domains mayform all or part of a polypeptide linker of a hybrid nuclease moleculeof the invention.

Preferred hybrid nuclease molecules of the invention comprise at leastone nuclease domain (e.g., RNase or DNase), at least one linker domain,and at least one Fc domain

In certain embodiments, the hybrid nuclease molecules of the inventionhave at least one nuclease domain specific for a target molecule whichmediates a biological effect. In another embodiment, binding of thehybrid nuclease molecules of the invention to a target molecule (e.g.DNA or RNA) results in the reduction or elimination of the targetmolecule, e.g., from a cell, a tissue, or from circulation.

In certain embodiments, the hybrid nuclease molecules of the inventionmay comprise two or more nuclease domains. In one embodiment, thenuclease domains are identical, e.g., RNase and RNase, or TREX1 andTREX1. In another embodiment, the nuclease domains are different, e.g.,DNase and RNase.

In other embodiments, the hybrid nuclease molecules of the invention maybe assembled together or with other polypeptides to form bindingproteins having two or more polypeptides (“multimers”), wherein at leastone polypeptide of the multimer is a hybrid nuclease molecule of theinvention. Exemplary multimeric forms include dimeric, trimeric,tetrameric, and hexameric altered binding proteins and the like. In oneembodiment, the polypeptides of the multimer are the same (ie. homomericaltered binding proteins, e.g. homodimers, homotetramers). In anotherembodiment, the polypeptides of the multimer are different (e.g.heteromeric).

Methods of Making Hybrid Nuclease Molecules

The hybrid nuclease molecules of this invention largely may be made intransformed host cells using recombinant DNA techniques. To do so, arecombinant DNA molecule coding for the peptide is prepared. Methods ofpreparing such DNA molecules are well known in the art. For instance,sequences coding for the peptides could be excised from DNA usingsuitable restriction enzymes. Alternatively, the DNA molecule could besynthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused.

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of affecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal nuclease domains, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257-527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides. Compounds that contain derivatized peptides orwhich contain non-peptide groups may be synthesized by well-knownorganic chemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

Pharmaceutical Compositions and Therapeutic Methods of Use

In certain embodiments, a hybrid nuclease molecule is administeredalone. In certain embodiments, a hybrid nuclease molecule isadministered prior to the administration of at least one othertherapeutic agent. In certain embodiments, a hybrid nuclease molecule isadministered concurrent with the administration of at least one othertherapeutic agent. In certain embodiments, a hybrid nuclease molecule isadministered subsequent to the administration of at least one othertherapeutic agent. In other embodiments, a hybrid nuclease molecule isadministered prior to the administration of at least one othertherapeutic agent. As will be appreciated by one of skill in the art, insome embodiments, the hybrid nuclease molecule is combined with theother agent/compound. In some embodiments, the hybrid nuclease moleculeand other agent are administered concurrently. In some embodiments, thehybrid nuclease molecule and other agent are not administeredsimultaneously, with the hybrid nuclease molecule being administeredbefore or after the agent is administered. In some embodiments, thesubject receives both the hybrid nuclease molecule and the other agentduring a same period of prevention, occurrence of a disorder, and/orperiod of treatment.

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. In certainembodiments, the combination therapy comprises nuclease molecule, incombination with at least one other agent. Agents include, but are notlimited to, in vitro synthetically prepared chemical compositions,antibodies, antigen binding regions, and combinations and conjugatesthereof. In certain embodiments, an agent can act as an agonist,antagonist, allosteric modulator, or toxin.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising a hybrid nuclease molecule together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising a hybrid nuclease molecule and a therapeuticallyeffective amount of at least one additional therapeutic agent, togetherwith a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Insome embodiments, the formulation material(s) are for s.c. and/or I.V.administration. In certain embodiments, the pharmaceutical compositioncan contain formulation materials for modifying, maintaining orpreserving, for example, the pH, osmolarity, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. In certain embodiments,suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HC1, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company (1995). In some embodiments, the formulationcomprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH5.2, 9% Sucrose.

In certain embodiments, a hybrid nuclease molecule and/or a therapeuticmolecule is linked to a half-life extending vehicle known in the art.Such vehicles include, but are not limited to, polyethylene glycol,glycogen (e.g., glycosylation of the hybrid nuclease molecule), anddextran. Such vehicles are described, e.g., in U.S. application Ser. No.09/428,082, now U.S. Pat. No. 6,660,843 and published PCT ApplicationNo. WO 99/25044, which are hereby incorporated by reference for anypurpose.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. In someembodiments, the saline comprises isotonic phosphate-buffered saline. Incertain embodiments, neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. In certain embodiments,pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, oracetate buffer of about pH 4.0-5.5, which can further include sorbitolor a suitable substitute therefore. In certain embodiments, acomposition comprising a hybrid nuclease molecule, with or without atleast one additional therapeutic agents, can be prepared for storage bymixing the selected composition having the desired degree of purity withoptional formulation agents (Remington's Pharmaceutical Sciences, supra)in the form of a lyophilized cake or an aqueous solution. Further, incertain embodiments, a composition comprising a hybrid nucleasemolecule, with or without at least one additional therapeutic agent, canbe formulated as a lyophilizate using appropriate excipients such assucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desired hybridnuclease molecule, with or without additional therapeutic agents, in apharmaceutically acceptable vehicle. In certain embodiments, a vehiclefor parenteral injection is sterile distilled water in which a hybridnuclease molecule, with or without at least one additional therapeuticagent, is formulated as a sterile, isotonic solution, properlypreserved. In certain embodiments, the preparation can involve theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that canprovide for the controlled or sustained release of the product which canthen be delivered via a depot injection. In certain embodiments,hyaluronic acid can also be used, and can have the effect of promotingsustained duration in the circulation. In certain embodiments,implantable drug delivery devices can be used to introduce the desiredmolecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, a hybrid nuclease molecule, withor without at least one additional therapeutic agent, can be formulatedas a dry powder for inhalation. In certain embodiments, an inhalationsolution comprising a hybrid nuclease molecule, with or without at leastone additional therapeutic agent, can be formulated with a propellantfor aerosol delivery. In certain embodiments, solutions can benebulized. Pulmonary administration is further described in PCTapplication no. PCT/US94/001875, which describes pulmonary delivery ofchemically modified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, a hybrid nuclease molecule,with or without at least one additional therapeutic agents, that isadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized In certain embodiments, at leastone additional agent can be included to facilitate absorption of ahybrid nuclease molecule and/or any additional therapeutic agents. Incertain embodiments, diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of a hybrid nuclease molecule, with or without atleast one additional therapeutic agents, in a mixture with non-toxicexcipients which are suitable for the manufacture of tablets. In certainembodiments, by dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions can be prepared in unit-dose form. Incertain embodiments, suitable excipients include, but are not limitedto, inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving a hybrid nuclease molecule,with or without at least one additional therapeutic agent(s), insustained- or controlled-delivery formulations. In certain embodiments,techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci.USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising a hybrid nuclease molecule, with or without atleast one additional therapeutic agent, to be employed therapeuticallywill depend, for example, upon the therapeutic context and objectives.One skilled in the art will appreciate that the appropriate dosagelevels for treatment, according to certain embodiments, will thus varydepending, in part, upon the molecule delivered, the indication forwhich a hybrid nuclease molecule, with or without at least oneadditional therapeutic agent, is being used, the route ofadministration, and the size (body weight, body surface or organ size)and/or condition (the age and general health) of the patient. In certainembodiments, the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. In certainembodiments, a typical dosage can range from about 0.1 μg/kg to up toabout 100 mg/kg or more, depending on the factors mentioned above. Incertain embodiments, the dosage can range from 0.1 μg/kg up to about 100mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of a hybrid nuclease molecule and/or anyadditional therapeutic agents in the formulation used. In certainembodiments, a clinician will administer the composition until a dosageis reached that achieves the desired effect. In certain embodiments, thecomposition can therefore be administered as a single dose, or as two ormore doses (which may or may not contain the same amount of the desiredmolecule) over time, or as a continuous infusion via an implantationdevice or catheter. Further refinement of the appropriate dosage isroutinely made by those of ordinary skill in the art and is within theambit of tasks routinely performed by them. In certain embodiments,appropriate dosages can be ascertained through use of appropriatedose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it can be desirable to use a pharmaceuticalcomposition comprising a hybrid nuclease molecule, with or without atleast one additional therapeutic agent, in an ex vivo manner In suchinstances, cells, tissues and/or organs that have been removed from thepatient are exposed to a pharmaceutical composition comprising a hybridnuclease molecule, with or without at least one additional therapeuticagent, after which the cells, tissues and/or organs are subsequentlyimplanted back into the patient.

In certain embodiments, a hybrid nuclease molecule and/or any additionaltherapeutic agents can be delivered by implanting certain cells thathave been genetically engineered, using methods such as those describedherein, to express and secrete the polypeptides. In certain embodiments,such cells can be animal or human cells, and can be autologous,heterologous, or xenogeneic. In certain embodiments, the cells can beimmortalized. In certain embodiments, in order to decrease the chance ofan immunological response, the cells can be encapsulated to avoidinfiltration of surrounding tissues. In certain embodiments, theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

The hybrid nuclease molecules of the instant invention are particularlyeffective in the treatment of autoimmune disorders or abnormal immuneresponses. In this regard, it will be appreciated that the hybridnuclease molecules of the present invention may be used to control,suppress, modulate, treat, or eliminate unwanted immune responses toboth external and autoantigens. In yet other embodiments thepolypeptides of the present invention may be used to treat immunedisorders that include, but are not limited to, insulin-dependentdiabetes mellitus, multiple sclerosis, experimental autoimmuneencephalomyelitis, rheumatoid arthritis, experimental autoimmunearthritis, myasthenia gravis, thyroiditis, an experimental form ofuveoretinitis, Hashimoto's thyroiditis, primary myxoedema,thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis,Addison's disease, premature menopause, male infertility, juvenilediabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid,sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolyticanaemia, idiopathic leucopenia, primary biliary cirrhosis, activechronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis,Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis,dermatomyositis, discoid LE, systemic lupus erythematosus, or connectivetissue disease.

Kits

A kit can include a hybrid nuclease molecule disclosed herein andinstructions for use. The kits may comprise, in a suitable container, ahybrid nuclease molecule disclosed herein, one or more controls, andvarious buffers, reagents, enzymes and other standard ingredients wellknown in the art.

The container can include at least one vial, well, test tube, flask,bottle, syringe, or other container means, into which a hybrid nucleasemolecule may be placed, and in some instances, suitably aliquoted. Wherean additional component is provided, the kit can contain additionalcontainers into which this component may be placed. The kits can alsoinclude a means for containing the hybrid nuclease molecule and anyother reagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained. Containers and/or kits can includelabeling with instructions for use and/or warnings.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: MackPublishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry3^(rd) Ed. (Plenum Press) Vols A and B(1992).

Example 1 General Approach for Generating Hybrid Nuclease Molecules

Hybrid nuclease molecules were designed to incorporate desiredstructures and functional activity of single enzyme or multi-enzymestructures as modular cassettes with compatible restriction enzyme sitesfor shuttling and domain exchange. The schematic structure of differentembodiments of hybrid nuclease molecules is illustrated in FIG. 1. Thenucleotide and amino acid sequences of representative hybrid nucleasemolecules are shown in Table 1.

Human cDNAs were isolated from human pancreas RNA (Ambion) or human PBMCRNA from normal human peripheral blood lymphocytes (approximately5×10e6) using QIAgen RNAeasy kits (Valencia, Calif.) and QIAshredderkits to homogenize cell lysates (Qiagen, Valencia, Calif.). Human PBMCswere isolated from heparinized human blood diluted 1:1 in D-PBS andlayered over LSM Lymphocyte Separation Medium (MP Biomedicals, Irvine,Calif.) Ficoll gradients.

Mouse spleen RNA was isolated using QIAgen RNAeasy kits (Valencia,Calif.) from approximately 5×10e6 splenocytes. Cells were pelleted bycentrifugation from the culture medium, and 5×10e6 cells were used toprepare RNA. RNA was isolated from the cells using the QIAGEN RNAeasykit (Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredderaccording to the manufacturer's instructions accompanying the kit. Oneto two microgram (1-2 μg) of total RNA was used as template to preparecDNA by reverse transcription. The RNA, 300 ng random primers, and 500ng Oligo dT (12-18), and 1 μl 25 mM dNTPs were combined and denatured at80° C. for 5 minutes prior to addition of enzyme. Superscript IIIreverse transcriptase (Invitrogen, Life Technologies) was added to theRNA plus primer mixture in a total volume of 25 μl in the presence of 5times second strand buffer and 0.1 M DTT provided with the enzyme. Thereverse transcription reaction was allowed to proceed at 50° C. for onehour.

Between 10-100 ng cDNA was used in PCR amplification reactions usingprimers specific for the nuclease gene of interest (RNaseA, RNase1,DNase1, Trex1, DNase1L3, etc.) For initial cloning reactions, primerswere designed to isolate the full length cDNA or truncation productsencoding the gene of interest. Full length or shortened PCR fragmentswere isolated by agarose gel electrophoresis, and purified using QiagenQlAquick columns to remove nucleotides, primers, and unwanted amplifiedproducts. Purified fragments were cloned into pCR2.1 TOPO cloningvectors (Invitrogen, Carlsbad, Calif.) and transformed into TOP10competent bacteria. Isolated colonies were picked into Luria Broth mediacontaining 50 ug/ml carbenicillin, and grown overnight to isolateplasmids. TOPO clones were screened for inserts of the correct size bydigestion with EcoRI (NEB, Ipswich, Mass.) restriction enzyme andagarose gel electrophoresis of digested fragments. DNA sequence analysisof positive clones was performed with ABI Ready Reaction Mix v 3.1 andanalyzed using an ABI 3730 XL DNA sequencer. Once correct clones wereobtained, further sequence modifications were designed and PCR reactionsperformed to generate the desired alleles or expression cassettes.Truncation products and alleles were generated by PCR mutagenesis usingoverlapping primers for introduction of mutations at specific positionsin the genes. Linkers were synthesized by overlapping PCR using internaloverlapping primers and successive rounds of PCR to attach additionalsequence to each terminus. Hybrid nuclease molecules were assembled as astring of several interchangeable cassettes. Molecules of the preferredembodiment contain a fixed leader peptide, a nuclease cassette, anoptional cassette encoding a choice of several different polypeptidelinkers, an −Ig Fc domain cassette with either a STOP codon or a linkerat the carboxyl end of the CH3 domain, and for resolvICase typemolecules, a second linker cassette, followed by a second nucleasecassette. FIG. 1 illustrate the cassette type structure of these hybridnuclease molecules and examples of potential sequences inserted at eachposition. Once hybrid nuclease molecules were assembled, they weretransferred to a mammalian expression plasmid pDG appropriate fortransient expression in COS7 or other cells and stable expression in CHODG44 cells using selection for DHFR with methotrexate.

Transient Expression of Hybrid Nuclease Molecules

COS-7 cells were transiently transfected with expression vector pDGcontaining hybrid nuclease molecule gene inserts. The day beforetransfection, cells were seeded at 4×10e5 cells per 60 mm dish in 4 mlDMEM (ThermoFisher/Mediatech cell gro)+10% FBS tissue culture media.DMEM basal media was supplemented with 4.5 g/L glucose, sodium pyruvate,L-glutamine 4 mM, and non-essential amino acids. Fetal bovine serum(Hyclone, Logan, UT ThermoFisher Scientific) was added to media at 10%final volume. Cells were incubated at 37° C., 5% CO2 overnight and wereapproximately 40-80% confluent on the day of transfection. Plasmid DNAwas prepared using Qiagen (Valencia, Calif.) QIAprep miniprep kitsaccording to manufacturer's instructions, and eluted in 50 ul EB buffer.DNA concentrations were measured using a Nanodrop 1000 (Thermo FisherScientific, Wilmington Del.) spectrophotometer. Plasmid DNA wastransfected using Polyfect (Qiagen, Valencia, Calif.) transfectionreagent according to manufacturer's instructions, using 2.5 ug plasmidDNA per 60 mm dish and 15 ul polyfect reagent in 150 ul serum free DMEMtransfection cocktails. After complex formation, reactions were dilutedinto 1 ml cell growth media containing serum and all supplements, andadded drop-wise to the plates containing 3 ml fresh DMEM completeculture media. Transient transfections were incubated for 48-72 hoursprior to harvesting culture supernatants for further analysis.

Generation of Stable CHO DG44 Transfectants Expressing the HybridNuclease Molecules of Interest

Stable production of the hybrid nuclease molecules was achieved byelectroporation of a selectable, amplifiable plasmid, pDG, containingthe nuclease-Ig cDNA under the control of the CMV promoter, into ChineseHamster Ovary (CHO) cells. The pDG vector is a modified version ofpcDNA3 encoding the DHFR selectable marker with an attenuated promoterto increase selection pressure for the plasmid. Plasmid DNA was preparedusing Qiagen maxiprep kits, and purified plasmid was linearized at aunique Ascl site prior to phenol extraction and ethanol precipitation.Salmon sperm DNA (Sigma-Aldrich, St. Louis, Mo.) was added as carrierDNA, and 100 μg each of plasmid and carrier DNA was used to transfect10⁷ CHO DG44 cells by electroporation. Cells were grown to logarithmicphase in Excell 302 media (JRH Biosciences) containing glutamine (4 mM),pyruvate, recombinant insulin, penicillin-streptomycin, and 2× DMEMnonessential amino acids (all from Life Technologies, Gaithersburg,Md.), hereafter referred to as “Excell 302 complete” media. Media foruntransfected cells also contained HT (diluted from a 100× solution ofhypoxanthine and thymidine) (Invitrogen/Life Technologies). Media fortransfections under selection contained varying levels of methotrexate(Sigma-Aldrich) as selective agent, ranging from 50 nM to 1 μM.Electroporations were performed at 280 volts, 950 microFarads.Transfected cells were allowed to recover overnight in non-selectivemedia prior to selective plating in 96 well flat bottom plates (Costar)at varying serial dilutions ranging from 125 cells/well to 2000cells/well. Culture media for cell cloning was Excell 302 complete,containing 50 nM methotrexate. Once clonal outgrowth was sufficient,serial dilutions of culture supernatants from master wells were screenedfor expression of hybrid nuclease molecules by use of an −IgG sandwichELISA. Briefly, NUNC immulon II plates were coated overnight at 4° C.with 7.5 microgram/ml F(ab′2) goat anti-mouse IgG (KPL Labs,Gaithersburg, Md.) or 2 ug/ml goat anti-human or anti-mouse IgG (JacksonImmunoresearch, West Grove Pa.) in PBS. Plates were blocked in PBS/2-3%BSA, and serial dilutions of culture supernatants incubated at roomtemperature for 2-3 hours. Plates were washed three times in PBS/0.05%Tween 20, and incubated with horseradish peroxidase conjugatedF(ab′2)goat anti-mouse IgG2a (Southern Biotechnologies) and goatanti-mouse IgG (KPL) mixed together, each at 1:3500 in PBS/1.0% BSA, orin horseradish peroxidase conjugated F(ab′)2 goat anti-human IgG1(Jackson Immunoresearch, West Grove, Pa.) at 1:2500 for 1-2 hours atroom temperature. Plates were washed four times in PBS/0.05% Tween 20,and binding detected with SureBlue Reserve, TMB substrate (KPL Labs,Gaithersburg, Md.). Reactions were stopped by addition of equal volumeof 1N HCl, and plates read at 450 nM on a Spectramax Pro plate reader(Microdevices, Sunnyvale Calif.). The clones with the highest productionof the hybrid nuclease molecule were expanded into T25 and then T75flasks to provide adequate numbers of cells for freezing and for scalingup production of the fusion protein. Production levels were furtherincreased in cultures from the four best clones by progressiveamplification in methotrexate containing culture media. At eachsuccessive passage of cells, the Excell 302 complete media contained anincreased concentration of methotrexate, such that only the cells thatamplified the DHFR plasmid could survive.

Supernatants were collected from CHO cells expressing the hybridnuclease molecule, filtered through 0.2 μm PES express filters (Nalgene,Rochester, N.Y.) and were passed over a Protein A-agarose (IPA 300crosslinked agarose) column (Repligen, Needham, Mass.). The column waswashed with column wash buffer (90 mM Tris-Base, 150 mM NaCl, 0.05%sodium azide, pH 8.7), and bound protein was eluted using 0.1 M citratebuffer, pH 3.0. Fractions were collected and protein concentration wasdetermined at 280 nM using a Nanodrop (Wilmington Del.) microsamplespectrophotometer, and blank determination using 0.1 M citrate buffer,pH 3.0. Fractions containing hybrid nuclease molecules were pooled, andbuffer exchange performed by serial spins in PBS using centriconconcentrators followed by filtration through 0.2 μm filter devices, toreduce the possibility of endotoxin contamination.

Example 2 Construction of RNase-Ig Fusion Genes

Murine RNase 1 was amplified as a full-length cDNA from an EST library(from Dr. C. Raine, Albert Einstein School of Medicine, Bronx, N.Y.) whosent the clone to our laboratory without an MTA. Sequence specific 5′and 3′ primers used were from the published sequences. The sequence ofthe clone was verified by sequencing analysis. The Genebank accessionnumber is NCBI geneID 19752. Full length human RNase 1 was isolated fromrandom primed and oligo dT primed cDNA derived from human pancreas totalRNA (Ambion/Applied Biosystems, Austin, Tex.).

Once a full-length clone was isolated, primers were designed to create afusion gene with the mouse IgG2a or human IgG1 (SEQ ID NO:40) Fcdomains. Two different primers were designed for the 5′ sequence fusedat the amino terminus of the Fc tail; the first incorporated the nativeleader peptide from mouse (or human) RNase, while the second attached anAgel site to the amino terminus of RNase at the predicted signal peptidecleavage site in order to fuse the RNase to a human VKIII leader peptidethat we already had cloned and used for other expression studies. Forthe murine RNase, the sequence of the first primer is:

mribNL5′

30 mer (RNase 5′ with native leader and HindIII+Kozak)

(SEQ ID NO: 1) gTT AAg CTT gCC ACC ATg ggT CTg gAg AAg TCC CTCATT CTg-3′

The second primer creates a gene fusion junction between an existingleader sequence and the mature sequence at the 5′ end of the RNase, ator near the predicted leader peptide cleavage site.

27 mer (RNase 5′ mature sequence (no leader, with AgeI site)

(SEQ ID NO: 2) 5′-gAT ACC ACC ggT Agg gAA TCT gCA gCA CAg AAg TTT CAg-3′

The sequence of the 3′ primer for fusion to murine IgG2a at the carboxyend of RNase and the amino terminus of the Fc tail is as follows:

mrib3NH2

28 mer (RNase 3′ end with XhoI site for fusion to mIgG2a).

(SEQ ID NO: 3) 5′-ggC TCg AgC ACA gTA gCA TCA AAg tGG ACT ggT ACgTAg g-3′

Two more oligos were designed to create an −Ig-RNase fusion gene, wherethe −Ig tail is amino terminal to the RNase enzyme domain.

mrib5X

36 mer RNase 5′ end with linker aa and XbaI site for fusion to carboxyend of Fc domain

(SEQ ID NO: 4) 5′-AAA TCT AgA CCT CAA CCA ggT Agg gAA TCT gCA gCACAg AAg TTT CAg-3′

mrib3X

31mer RNase 3′ end with two stop codons and XbaI site for fusion tocarboxy end of Fc domain.

(SEQ ID NO: 5) 5′-TCT AgA CTA TCA CAC AgT AgC ATC AAA gTg gAC TggTAC gTA g-3′

Example 3 Isolation of Human and Mouse −Fc Domains and Introduction ofMutations into the Coding Sequence

For isolation of mouse and human −Fc domains (SEQ ID NO:40), RNA wasderived from mouse or human tissue as follows. A single cell suspensionwas generated from mouse spleen in RPMI culture media. Alternatively,human PBMCs were isolated from fresh, whole blood using LymphocyteSeparation Media (LSM) Organon Teknika (Durham, N.C.), buffy coatsharvested according to manufacturer's directions, and cells washed threetimes in PBS prior to use. Cells were pelleted by centrifugation fromthe culture medium, and 2×10⁷ cells were used to prepare RNA. RNA wasisolated from the cells using the QIAGEN RNAeasy kit (Valencia, Calif.)total RNA isolation kit and QIAGEN QIAshredder columns according to themanufacturer's instructions accompanying the kits. One microgram (4 μg)of total RNA was used as template to prepare cDNA by reversetranscription. The RNA, 300 ng random primers, and 500 ng Oligo dT(12-18), and 1 μl 25 mM dNTPs were combined and denatured at 80° C. for5 minutes prior to addition of enzyme. Superscript III reversetranscriptase (Invitrogen, Life Technologies) was added to the RNA plusprimer mixture in a total volume of 25 μl in the presence of .secondstrand buffer and 0.1 M DTT provided with the enzyme. The reversetranscription reaction was allowed to proceed at 50° C. for one hour.cDNA was purified using QlAquick (QIAGEN) PCR purification columnsaccording to manufacturer's directions, and eluted in 40 microliters EBbuffer prior to use in PCR reactions.

Wild type mouse and human −Fc domains were isolated by PCR amplificationusing the cDNA described above as template. The following primers wereused for initial amplification of wild type sequences, but incorporatedthe desired mutational changes in the hinge domain:

mahIgG1CH2M: 47 mer (SEQ ID NO: 6)5′-tgtccaccgtgtccagcacctgaactcctgggtggatcgtcagtctt cc-3′hIgG1-5scc: 49 mer (SEQ ID NO: 7)5′-agatctcgagcccaaatcttctgacaaaactcacacatgtccaccgt gt-3′mahIgG1S: 51 mer (SEQ ID NO: 8)5′-tctagattatcatttacccggagacagagagaggctcttctgcgtgt agtg-3′muIgG2aCH2: 58mer (SEQ ID NO: 9)5′-cctccatgcaaatgcccagcacctaacctcttgggtggatcatccgt cttcatcttcc-3′mIgG2a-5scc: 47mer (SEQ ID NO: 10)5′-gaagatctcgagcccagaggtcccacaatcaagccctctcctcca- 3′ mIgG2a3S: 48mer(SEQ ID NO: 11) 5′-gtttctagattatcatttacccggagtccgagagaagctcttagtc gt-3′

PCR reactions were performed using a C1000 thermal cycler (BioRad,Hercules Calif.) or an Eppendorf thermal cycler (ThermoFisherScientific, Houston Tex.). Reactions included an initial denaturationstep at 95° C. for 2 minutes, followed by 34 cycles with a 94° C., 30sec denaturation, 50° C., 30 sec annealing, and 72° C., 1 minuteextension step, followed by a final 4 minute extension at 72° C. Oncewild type tails were isolated, the fragments were TOPO cloned intopCR2.1 vectors, DNA prepared using the QIAGEN spin plasmid miniprep kitsaccording to manufacturer's instructions and clones sequenced using ABIDye Terminator v3.1 sequencing reactions according to manufacturer'sinstructions.

DNA from the correct clones were used as templates in overlap extensionPCRs to introduce mutations at the desired positions in the codingsequence for mouse IgG2a or human −IgG1. PCR reactions were set up usingthe full length wild type clones as template (1 microliter), 50 pmol 5′and 3′ primers to PCR each portion of the −Fc domain up to and includingthe desired mutation site from each direction, and PCR hi fidelitySupermix (Invitrogen, Carlsbad Calif.), in 50 microliter reactionvolumes using a short amplification cycle. As an example of theoverlapping PCR mutagenesis, the primer combination used to introducethe P331S mutation into human −IgG1, was as follows:

A 5′ subfragment was amplified using the full-length wild type clone astemplate, and the 5′ primer was hIgG1-5scc:5′-agatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgt-3′ (SEQ ID NO:12),while the 3′ primer was P331AS:5′-gttttctcgatggaggctgggagggctttgttggagacc-3′ (SEQ ID NO:13). A 3′subfragments was amplified using the full-length wild type clone astemplate and the 5′ primer was P331S:5′aaggtctccaacaaagccctcccagcctccatcgagaaaacaatctcc-3′ (SEQ ID NO:14),while the 3′ primer was mahIgG1S:5′-tctagattatcatttacccggagacagagagaggctcttctgcgtgtagtg-3′ (SEQ IDNO:15).

Once subfragments were amplified and isolated by agarose gelelectrophoresis, they were purified by QIAquick gel purification columnsand eluted in 30 microliters EB buffer according to manufacturer'sinstructions. Two rounds of PCR were then performed with the twosubfragments as overlapping templates in new reactions. The cycler waspaused and the 5′ (hIgG1-5scc, see above) and 3′ (mahIgG1S, see above)flanking primers were added to the reactions (50 pmol each). PCRamplifications were then carried out for 34 cycles at the conditionsdescribed for the wild type molecules above. Full length fragments wereisolated by gel electrophoresis, and TOPO cloned into pCR2.1 vectors forsequence analysis. Fragments from clones with the correct sequence werethen subcloned into expression vectors for creation of the differenthybrid nuclease molecules described herein.

Example 4 Quantitation of RSLV-124 Protein and RNase Enzyme Activity inMouse Sera

In Vivo Mouse Stability Analysis of RSLV-124 Construct (SEQ ID NO:106).

Four mice (C220, C221, C222, C223) were injected intravenously with asingle injection of RSLV-124 at time zero. At various times followingthe injection blood samples were collected and analyzed for the presenceof RSLV-124 protein (a human wild type RNase linked to a wild-type humanIgG1 Fc domain (SEQ ID NO:106)) and RNase enzymatic activity. To detectthe RSLV-124 compound in mouse serum, an ELISA was developed whichcaptures the human Fc from the mouse serum followed by detection of thehuman RNase. When the ELISA was run on the blood samples of the fourmice the presence of RSLV-124 protein was detected at five minutesfollowing a single intravenous injection of 150 ug, at between 38 μg/mlto 55 μg/ml (FIG. 2). At one day post injection the concentration ofRSLV-124 dropped rapidly to between 8 μg/ml to 12 μg/ml. The bloodconcentration of the drug remained relatively stable for the duration ofthe analysis out to seven days where blood levels of the drug wereapproximately 5 μg/ml. The same blood samples used to measure theRSLV-124 protein by ELISA were used to quantitate the RNase enzymaticactivity of the drug. The RNaseAlert QC system from Ambion (Cat #AM1966)was used to measure the enzyme kinetics of the RSLV-124 protein in mouseblood samples with some modifications. The drug compound was capturedfrom mouse serum onto the RNaseAlert assay plate using a human anti-Fcmonoclonal antibody and quantitated by measurement of fluorescence perthe Ambion kit instructions. Analysis of the relative fluorescent units(RFU's) of the RSLV-124 molecule showed between 80,000-140,000 RFU's atfive minutes after injection (FIG. 3). The RFU's declined rapidly inparallel with the protein concentration, remaining relatively stable atbetween 18,000-40,000 RFU's out to day seven. The RNaseAlert QC systemwas used to develop a standard curve using known quantities of protein,from this standard curve the RFU's of the RSLV-124 in the blood sampleswere used to extrapolate the protein concentration of RSLV-124. Fromthis analysis it was determined that the protein concentration presentin the mouse blood over the seven day experiment as calculated using theRNase enzymatic activity assay were very similar to the values that weremeasured using the ELISA (FIG. 4). From these experiments it wasconcluded that the RSLV-124 compound is stable in vivo in mousecirculation over seven days and retains its enzymatic activity,suggesting that the compound is not susceptible to degradation in vivoin the mouse since nearly 100% of the enzymatic activity is retainedover seven days in the mouse circulation. Since Fc fusion proteins areoften susceptible to degradation in the circulation this finding furtherconfirms the use of the RNase-Fc fusion proteins as valuable drugs.

Example 5 Phenotype of TLR7.1xRNaseA Double Transgenic Mice

Mice were created that overexpress RNaseA (RNase Tg). This nuclease isexpressed at high levels in RNase Tg mice. We have developed both asingle radial diffusion (SRED) method (FIG. 5 and a much morequantitative ELISA to quantify RNase in the serum (FIG. 6). Both assaysshow a significant increase in RNase activity in the RNase Tg.Quantitation of the level of RNase in FIG. 6 compared with wild type B6mice showed there was an approximately 10-fold increase in RNase in theRNase Tg. We crossed RNaseA Tg with TLR7.1 Tg mice to create the doubleTg (DTg). TLR7.1 mice have 8-16 copies of TLR7 and develop a veryaggressive, rapidly progressive lupus-like disease and start to die at 3mo of age with a median survival of 6 mo. In a preliminary analysis, webled DTg and littermate controls at 3 mo of age to see whether the DTgmice exhibited signs of improvement. As shown in FIG. 5, DTg mice hadvery high levels of RNase in their serum (equivalent to >13 U/ml RNasebased on our standard with specific activity of 993 U/mg). RNaseAconcentration in Tg and DTg mice was also measured by ELISA assay asshown in FIG. 6. The RNase A Tg and TLR7.1XRNaseA Dtg mice have RNase Aserum concentrations between 1-2 ng/ml.

Detailed Method for Rnase A ELISA

-   -   1. Coat plate with anti-RnaseA Abcam Ab(ab6610): 2.5-10 ug/ml        O/N in 4 C.    -   2. Wash plate 3 times with 0.05% Tween/1× PBS    -   3. Block with 1% BSA in PBS for at least 1 hour    -   4. Wash plate 3 times with 0.05% Tween/1× PBS    -   5. Load samples. Sample dilutions at 1:50    -   6. Incubate Rm Temp for 2 hours    -   7. Wash plate 3 times with 0.05% Tween/1× PBS    -   8. Prepare dilution of biotin labeled Anti Rnase Ab at dilution        of 1:4500 (2.2 ug/ml). Leave RT for 1 hour (Rockland 200-4688:        10 mg/ml).    -   9. Wash plate 3 times    -   10. Dilute StrepAV HRP (Biolegend 405210) 1:2500. Cover with        foil and leave at RT for 25-30 min.    -   11. Wash 6 times, let the liquid sit in wells for at least 30        seconds in between washes.    -   12. Add BD OptEIA substrate A+B 1:1. Wait until color changes        5-10 min max. Don't let the top well standard go over 1.0. Add        80 ul. (CatNos: 51-2606KC; ReagentA, 51-2607KC; ReagentB)    -   13. Add 40 ul of 1M sulfuric acid to stop reaction

Product/Reagent Information:

RNaseA Ab: ab6610 (90 mg/ml)

ELISA buffer: 1% BSA in PBS

ELISA wash buffer: 0.05% Tween/1× PBS

Anti RNaseA biotin conjugated Ab: Rockland: 200-4688(10 mg/ml)

Strep AV HRP: Biolegend 405210

BD OptEIA reagent A and B: 51-2606KC and 51-2607KC

Example 6 Survival Curves for TLR7.1 Transgenic Mouse Strains

There was a highly significant difference between the DTg and the TLR7.1littermate controls in survival. As shown in FIG. 7, at 10 months, 61%of TLR7.1 mice had died, whereas 31% of DTg mice had died. This datashows that overexpression of RNaseA exerted a strong therapeutic effect.The reasons why TLR7.1 mice die prematurely is not entirely clear,although severe anemia, thrombocytopenia, and glomerulonephritis couldplay a part. To determine whether red cell and platelet counts werepositively impacted by RNaseA expression in the DTg mice, we performedblood counts but found no differences between the TLR7.1 and DTg mice.In contrast, there was a significant improvement in kidneyhistopathology in the DTg mice. We observed decreased deposition of IgGand C3 in DTg mice. PAS staining, which reflects inflammation in themesangium was also reduced in DTg mice compared to TLR7.1 littermatecontrols. When we have now compared macrophage infiltration of thekidneys using anti-MAC-2 (galectin3) antibody (Lyoda et al. Nephrol DialTransplat 22: 3451, 2007), there were many fewer mac-2 positive cells inthe glomeruli of the DTg mice. The results of counting 20 glomeruli permouse in 5 mice in each group revelaed mean+/−SE of 3.8+/−1.1 and1.4+/−0.2 for single versus DTg respectively, p=0.05. In addition, wequantified glomerular tuft size and observed a significant reduction inglomerular tuft size in the DTg mice (179+/−41 versus 128+/−16.8 um2 insingle versus DTg respectively, p=0.037). In summary, TLR7.1XRNaseA DTgmice survive longer than their single Tg TLR7.1 littermates and haveless inflammation and injury in their kidneys. This finding indicatesthat removing RNA immune complexes in this mouse model significantlyimproved overall mortality and decreased kidney damage and overallimflammation associated with this lupus-like pathology.

Example 7 Analysis of IRGs in Spleens of TLR Tg Mice

Analysis of interferon response genes (IRGs) in the spleens of TLR7.1 Tgand TLR7.1×RNaseA DTg mice mice showed that expression of the IRF7 gene(Interferon regulatory factor 7 (UniProtKB P70434)) was significantlylower in the DTg mice (p=0.03). Some other IRGs including MX1(Interferon-induced GTP-binding protein Mx1 (UniProtKB P09922)) and VIG1(Radical S-adenosyl methionine domain containing protein 2 (UniProtKBQ8CBB9)) were lower in DTg mice compared to Tg mice, but the differenceswere not significant. (FIG. 8). Quantitative PCR was performed asfollows: total RNA was isolated from mouse spleens using the RNeasy minikit (Qiagen, Valencia, Calif., USA), DNase treated using Turbo DNA-free(Applied Biosystems, Foster City, Calif., USA) and first-strand cDNA wasproduced with the RNA-to-cDNA kit (Applied Biosystems) using randomprimers. The 260/280 was between 1.7 and 2.0 for isolated RNA measuredwith a NanoDrop (Thermo Scientific, Waltham, Mass., USA). cDNA wasdiluted to an equivalent of 1 ng/ul total RNA and 8 ul were used perreaction. Primers for the reference gene (18 s) and genes of interest(GOI) were synthesized (IDT, Coralville, Iowa, USA) and diluted to theappropriate concentrations for qPCR using molecular grade water. BLASTresults of the primers show specific sequence homology only to thereference gene or GOI. Reactions in duplicate (20 ul) were run on an ABIFast 7500 system using a 1:1 mix of template and primer to SensiMix SYBRlow-ROX master mix (Bioline, London, UK). Relative quantification wascalculated using the 2^(−ddCT) method with age matched wild type B6 miceas baseline to determine fold changes for each GOI. The dissociationcurves for the reactions show a single melt peak for each gene. Thestandard curve showed similar amplification efficiencies for each geneand that template concentrations were within the linear dynamic rangefor each of primer set.

Example 8 Construction and Expression of DNase1-Ig Single and DualEnzyme Hybrid Nuclease Molecules

Naturally occurring alleles of human DNase1 or DNase1 like moleculeshave been reported. The A114F mutation has been previously reported tooccur in natural variants of human DNAsel like enzymes, and to result inactin resistance of the enzymes containing this sequence change. SeePan, C Q, Dodge T H, Baker D L, Prince W E, Sinicropi D V, and Lazarus RA. J Biol Chem 273: 18374-18381, (1998); Zhen A, Parmelee D, Hyaw H,Coleman T A, Su K, Zhang J, Gentz R, Ruben S, Rosen C, and Li Y. Biochemand Biophys Res Comm 231: 499-504 (1997); and Rodriguez A M, Rodin D,Nomura H, Morton C C, Weremowicz S, and Schneider M C. Genomics 42:507-513 (1997), all of which are herein incorporated by reference.

Similarly, the G105R mutation has been reported recently as a singlenucleotide polymorphism in the gene encoding human DNAse 1 that ispolymorphic in some or all populations, and that is relevant toautoimmunity. (See Yasuda T, Ueki M, Takeshita H, Fujihara J,Kimura-Kataoka K, Lida R, Tsubota E, Soejima M, Koda Y, Dato H, PanduroA. Int J Biochem Cell Biol 42(7): 1216-1225 (2010), herein incorporatedby reference). Allelic variants at this position resulted in highactivity harboring DNase 1 isoforms relative to wild type. Anothernaturally occurring, polymorphic mutation (R21S) has also been reportedto confer higher activity. (See Yasuda, supra)

SLE patients have been reported to have significantly decreased levelsof DNase1 activity (See Martinez-Valle F, Balada E, Ordi-Ros J,Bujan-Rivas S, Sellas-Fernandez A, Vilardell-Tarres M. Lupus 18(5):418-423 (2009), herein incorporated by reference).

Naturally occurring enzyme variants may thus be less immunogenic whenadministered to patients, since these isoforms occur in the humanpopulation. We reasoned that the combination of the actin resistantproperties of alleles similar to A114F with the increased enzymaticactivity of alleles like G105R would generate novel allelic variants ofhuman DNase1 that might show improved clinical activity in vitro and invivo. To our knowledge, ours is the first report of this new mutant formof DNase1 generated from a combination of two naturally occurringvariants G105R and A114F.

Human DNase 1 was isolated as described previously from human pancreasRNA (Ambion), by random primed cDNA and PCR using the following primersets:

5′hDNase1-age: (SEQ ID NO: 16)GTT ACC GGT CTG AAG ATC GCA GCC TTC AAC ATC CAG 5′hDNase1-bx:(SEQ ID NO: 17) GTT CTC GAG ATC TTT CAG CAT CAC CTC CAC TGG ATA GTG

Alternatively, the 3′ DNase cassettes were amplified by PCR using thefollowing primer pair.

3′hDNase1-RV: (SEQ ID NO: 18)GTT GAT ATC CTG AAG ATC GCA GCC TTC AAC ATC CAG 3′hDNase1-stop:(SEQ ID NO: 19) GTT TCT AGA TTA TCA CTT CAG CAT CAC CTC CAC TGG ATA GTG

PCR reactions were performed using 50 pmol each primer, 2 ul cDNA, in atotal volume of 50 ul using Platinum PCR Supermix as previouslydescribed. The amplification profile was 94 C 30 sec; 55 C 30 sec; 68 C90 sec for 35 cycles.

Once the wild type gene was amplified by PCR, the fragments weresubjected to gel electrophoresis and 850 bp fragments purified byQIAquick column purification. Fragments were cloned into pCR2.1,transformed by TOPO cloning according to manufacturer's instructions asdescribed for the other constructs. Once sequence was verified, PCRprimers were used to generate subfragments containing naturallyoccurring alleles for DNase1 that have been reported to improve specificactivity and improve resistance to the inhibitory activity of actin.These subfragments contained overlapping sequence, permittingamplification of complete DNase1 subclones containing the desiredallelic variations. COS 7 cells were transiently transfected in 60 mmdishes using Polyfect (Qiagen, Valencia, Calif.) transfection reagent.Plasmid DNA was prepared using the Qiagen QlAprep miniprep kitsaccording to manufacturer's instructions. Plasmids were eluted in 50 ulEB buffer. DNA concentration was measured using the Nanodrop and analiquot equivalent to 2.5 ug plasmid DNA used for each transfectionreaction. Each DNaseIg) or RNase-Ig-DNase) expression cassette wasinserted into the mammalian expression vector pDG, a derivative ofpcDNA3.1. Transfected cells were incubated for 72 hours at 37° C., 5%CO2 prior to harvest of culture supernatants for further analysis.Culture supernatants were harvested, residual cells centrifuged from thesolution, and the liquid transferred to new tubes. COS-7 cells weretransiently transfected with plasmids containing human DNase1 wild typeor naturally occurring DNase 1 mutant alleles (G105R and/or A114F))fused to the wild type human IgG1 Fc domain. This hinge-CH2-CH3 cassettecontains a single C→S mutation in the hinge region to eliminate thefirst cysteine in this domain since it is unpaired due to absence of itspairing partner present in the light chain of the antibody. In addition,more complex multi-nuclease fusion proteins were also expressed from COScell transient transfections.

Example 9 Isolation of Human −Ig Tails, Introduction of Mutations intothe Coding Sequence, and Construction of Mutant Nuclease Molecules

For isolation of mutant human −Ig Fc domains, RNA was derived from humanPBMCs isolated from fresh, whole blood using Lymphocyte Separation Media(LSM) Organon Teknika (Durham, N.C.), buffy coats harvested according tomanufacturer's directions, and cells washed three times in PBS prior touse. Cells were pelleted by centrifugation from the culture medium, and2×10₇ cells were used to prepare RNA. RNA was isolated from the cellsusing the QIAGEN RNAeasy kit (Valencia, Calif.) total RNA isolation kitand QIAGEN QIAshredder columns according to the manufacturer'sinstructions accompanying the kits. One microgram (4 μg) of total RNAwas used as template to prepare cDNA by reverse transcription. The RNA,300 ng random primers, and 500 ng Oligo dT (12-18), and 1 μl 25 mM dNTPswere combined and denatured at 80° C. for 5 minutes prior to addition ofenzyme. Superscript III reverse transcriptase (Invitrogen, LifeTechnologies) was added to the RNA plus primer mixture in a total volumeof 25 μl in the presence of second strand buffer and 0.1 M DTT providedwith the enzyme. The reverse transcription reaction was allowed toproceed at 50° C. for one hour. cDNA was purified using QIAquick(QIAGEN) PCR purification columns according to manufacturer'sdirections, and eluted in 40 microliters EB buffer prior to use in PCRreactions.

Wild type human −Ig Fc domains were isolated by PCR amplification usingthe cDNA described above as template. The mutant −Ig fragments wereisolated by PCR directed mutagenesis, using appropriate PCR primerscontaining the desired mutations and the wild type cassettes astemplate. PCR reactions were performed using a C1000 thermal cycler(BioRad, Hercules Calif.). Reactions included an initial denaturationstep at 95° C. for 2 minutes, followed by 34 cycles with a 94° C., 30second denaturation, 55° C., 30 second annealing, and 72° C., 1 minuteextension step, followed by a final 4 minute extension at 72° C. Oncefull length mutant tails were isolated, the fragments were TOPO clonedinto pCR2.1 vectors, DNA prepared using the QIAGEN spin plasmid miniprepkits according to manufacturer's instructions and clones sequenced usingABI Dye Terminator v3.1 sequencing reactions according to manufacturer'sinstructions.

Recombinant molecules were generated by PCR mutagenesis using overlapextension PCR with mutated oligonucleotides.

The following oligonucleotides were used to derive these molecules.

CS-P238S 5-1: (SEQ ID NO: 20)TCT CCA CCG AGC CCA GCA CCT GAA CTC CTG GGAGGA TCG TCA GTC TTC CTC TTC CCC C (58mer) SSSH-5-2: (SEQ ID NO: 21)AGA TCT CGA GCC CAA ATC TTC TGA CAA AAC TCA CACATC TCC ACC GAG CCC AGC ACC T (58 mer) P331S-S: (SEQ ID NO: 22)GTC TCC AAC AAA GCC CTC CCA GCC TCC ATC GAG AAA ACC ATC TCC A (46mer)P331S-AS: (SEQ ID NO: 23)TGG AGA TGG TTT TCT CGA TGG GGG CTG GGA GGG CTT TGT TGG AGA CC (47mer)hIgG1-3′WTnogt: (SEQ ID NO: 24)TCT AGA TTA TCA TTT TCC CGG AGA GAG AGA GAGGCT CTT CTG CGT GTA GTG (51mer)

The P238S mutation and SSS substitutions for SCC were introduced by PCRmutagenesis using two overlapping 5′ oligos in sequential PCR reactions.The first PCR reaction included the following 5′ primer thatincorporates the P238S mutation within its sequence: CS-P238S 5-1: TCTCCA CCG AGC CCA GCA CCT GAA CTC CTG GGA GGA TCG TCA GTC TTC CTC TTC CCCC (58 mer). (SEQ ID NO: 25)

The second PCR reaction included the following 5′ primer that overlappedthe first primer and added on the mutated hinge residues to the P238Smutant: SSSH-5-2: AGA TCT CGA GCC CAA ATC TTC TGA CAA AAC TCA CAC ATCTCC ACC GAG CCC AGC ACC T (58 mer). (SEQ ID NO: 26)

DNA from the correct clones was used as template in overlap extensionPCRs to introduce mutations at the desired internal positions in thecoding sequence for human −IgG1. PCR reactions were set up using thefull length clones as template (1 microliter), 50 pmol 5′ and 3′ primersto PCR each portion of the −Ig tail up to and including the desiredmutation site from each direction, and PCR hi fidelity Supermix(Invitrogen, Carlsbad Calif.), in 50 microliter reaction volumes using ashort amplification cycle. As an example of the overlapping PCRmutagenesis, the primer combination used to introduce the P331S mutationinto the human −IgG1 with the already introduced P238S mutation was asfollows:

A 5′ subfragment was amplified using the full-length wild type clone astemplate, and the 5′ primer was SSSH-5-2: AGA TCT CGA GCC CAA ATC TTCTGA CAA AAC TCA CAC ATC TCC ACC GAG CCC AGC ACC T (58 mer), while the 3′primer was P331S-AS: TGG AGA TGG TTT TCT CGA TGG GGG CTG GGA GGG CTT TGTTGG AGA CC (47 mer). (SEQ ID NO: 27)

A 3′ subfragments was amplified using the full length wild type clone astemplate and the 5′ primer: P331S-S: GTC TCC AAC AAA GCC CTC CCA GCC TCCATC GAG AAA ACC ATC TCC A (46 mer), while the 3′ primer washIgG1-3′WTnogt: TCT AGA TTA TCA TTT TCC CGG AGA GAG AGA GAG GCT CTT CTGCGT GTA GTG (51 mer). (SEQ ID NO: 28)

Once subfragments were amplified and isolated by agarose gelelectrophoresis, they were purified by QIAquick gel purification columnsand eluted in 30 microliters EB buffer according to manufacturer'sinstructions. Two rounds of PCR were then performed with the twosubfragments as overlapping templates in new reactions. The cycler waspaused and the 5′ and 3′ flanking primers were added to the reactions(50 pmol each). PCR amplifications were then carried out for 34 cyclesat the conditions described for the wild type molecules above. Fulllength fragments were isolated by gel electrophoresis, and TOPO clonedinto pCR2.1 vectors for sequence analysis. Fragments from clones withthe correct sequence were then subcloned into expression vectors forcreation of the different nuclease molecules described herein.

For multispecific nuclease molecules, PCR reactions were performed usingan alternative primer for the 3′ end of the Fc domain, removing the STOPcodon and adding on the NLG linker and the EcoRV restriction site to themolecules to facilitate fusion to the rest of the cassettes. The primersequence is listed below: 5′ GAT ATC CTG CAC GCT AGG GCT GCT CAC ATT 3′.(SEQ ID NO: 29)

RSLV mutant nucleases were constructed by fusing the mutated human −Igtails to the wild type RNase domain with or without a linker separatingthe two domains. RSLV 125 and RSLV126 fuse human RNase to the mutanthinge and IgG1 Fc domain. RSLV125 contains no linker, while RSLV 126contains the (gly4ser)4 linker as a (BglII-XhoI) fragment insertedbetween the nuclease domain and hinge regions. RSLV-125 incorporates awild type RNase cassette fused directly to an SSS (rather than CCC orwild type) version of the human IgG1 hinge, and a P238S, P331S mutanthuman IgG1 Fc domain (SEQ ID NO:61-62).

RSLV 126 incorporates a wild type RNase cassette fused to a (gly4ser)4linker domain, followed by the SSS mutant hinge, and a P238S-P331Sdouble mutant Fc domain (SEQ ID NO:63-64).

RSLV-127 is a multinuclease fusion construct that incorporates an aminoterminal human DNase (G105R/A114F) fused to a (gly4ser)4 linker domain,followed by an SSS mutant hinge and P238S-P331S double mutant Fc domainfused to an NLG linker domain and followed by a C terminal wild typeRNase domain (SEQ ID NO:65-66).

RSLV-128 is a multinuclease fusion construct that incorporates an aminoterminal wild type human RNase domain, fused to a (gly4ser)4 linkerdomain, followed by an SSS mutant hinge and P238S-P331S double mutant Fcdomain fused to an NLG linker domain and followed by a C terminal mutantDNase (G105R/A114F) domain (SEQ ID NO:67-68).

RSLV-129 is a multispecific fusion construct that incorporates an aminoterminal wild type human RNase domain, fused to an SSS mutant hinge andP238S-P331S double mutant Fc domain fused to an NLG linker domain andfollowed by a C terminal mutant DNase (G105R/A114F) domain (SEQ IDNO:69-70).

RSLV-132 incorporates a wild type RNase cassette fused directly to anSCC version of the human IgG1 hinge, and a P238S, P331S mutant humanIgG1 Fc domain (SEQ ID NOS: 91-92 and 95-96).

RSLV-133 is a multispecific fusion construct that incorporates an aminoterminal wild type human RNase domain, fused to an SCC mutant hinge andP238S-P331S double mutant Fc domain fused to an NLG linker domain andfollowed by a C terminal mutant DNase (G105R/A114F) domain (SEQ ID NOS:93-94 and 97-98).

Additional versions of RSLV-125-RSLV-129 with an SCC hinge are shown inTable 1 as RSLV-125-2 (SEQ ID NO:77-78), RSLV-126-2 (SEQ ID NO:79-80),RSLV-127-2 (SEQ ID NO:81-82), RSLV-128-2 (SEQ ID NO:83-84), andRSLV-129-2 (SEQ ID NO:85-86).

Example 10 Western Blot on RSLV 125-129 Fusion Proteins Expressed fromCOS7 Transfections

FIG. 9 shows a Western Blot on COS transfection supernatants from RSLV125-129 constructs. Expression plasmids containing RSLV 125, 126, 127,128, or 129 were transfected into COS7 cells using Polyfect transfectionreagent and supernatants harvested after 48 hours. In addition to thesingle nuclease molecules contained in RSLV 125 and 126, more complexmulti-nuclease fusion proteins were also expressed from COS celltransient transfections, encoded by RSLV 127, 128, and 129. Western blotanalysis was performed on supernatants from transient transfectants. Themolecules shown in FIG. 9 contain human RNase1 fused to the human SSSIgG1 hinge, and Ig G1 P238S-P331S Fc domain or include human RNase1(wild type) fused to the SSS hinge-(P238S-3315) CH2-CH3 Fc domain ofhuman IgG1, followed by a novel linker containing an N-linkedglycosylation site to protect the linker domain from protease cleavage,and the mutant allele G105R-A114F form of human DNase1 at the carboxyterminus of the molecule. In addition, RSLV 127 encodes the human DNase1mutant above at the amino terminus and the RNase 1 WT at the carboxyterminus of the mutant −Ig tail. COS supernatants were harvested after72 hours and 0.5 ml samples were immunoprecipitated overnight at 4° C.with 100 ul protein A-agarose beads. Protein A beads were centrifugedand washed twice in PBS prior to resuspending in SDS-PAGE loadingbuffer, for NuPAGE gels-reducing or nonreducing LDS sample buffer(Invitrogen, Carlsbad, Calif.). Samples were heated according tomanufacturer's instructions, protein A beads centrifuged to pellet, andsample buffer loaded onto 5-12% NuPAGE gradient gels. Samples wereelectrophoresed at 150 volts for 1.5-2 hours, and gels blotted tonitrocellulose membranes at 30 mAmp for 1 hour. Western blots wereblocked in TBS/5% non-fat milk overnight. Blots were incubated with1:2500 HRP (horseradish peroxidase) conjugated goat anti-human IgG (Fcspecific, Jackson Immunoresearch) for 1.5 hours at room temperature,washed in PBS/0.5% Tween20 five or more times, and blots developed usingECL reagent. The results demonstrate that the construction of thenuclease Fc fusion proteins was successful and the proteins are readilyexpressed from COS cells. Furthermore analyzing the reducing andnon-reducing profiles for these nuclease Fc fusion proteins confirmsthat the DNA constructs encode proteins of the appropriate molecularweight. The pattern on non-redcing SDS-PAGE confirms the di-sulfidebonding properties of the proteins are consistent with the expectedbehavior of the constructs.

Example 11 SRED Analysis of Affinity Purified Proteins from COS7Transfectants

FIG. 10 shows SRED analysis comparing aliquots of protein A purifiedproteins from RSLV transfected COS supernatants from Example 10. A 2%agarose gel was prepared with distilled water. Poly-IC (Sigma) wasdissolved in distilled water at 3 mg/ml. The gel plate was prepared asfollows: 1.5 ml reaction buffer (0.2M Tris-HCl pH 7.0, 40 mM EDTA and0.1 mg/ml ethidium bromide), 1 ml Poly-IC and 0.5 ml water were place inthe tube and maintained at 50° C. for 5 min. 3 ml of the agarose (keptat 50° C.) was added to the tube. The mixture was immediately pouredonto a glass plate. Sampling wells were punched in the gel. 2 μl of eachcontrol, serum sample, or affinity purified RSLV proteins, was loadedinto wells and the gel was incubated at 37° C. for 4 hours in the moistchamber. Then the gel was incubated in a buffer (20 mM sodium acetatepH5.2, 20 mg/ml ethidium bromide) on ice for 30 min. and read under UV.Gels were photographed on a UV transilluminator using a Kodak digitalcamera DC290 system equipped with ethidium bromide filters and analyzdusing Kodak Molecular Imaging software. The results from the RNaseenzymatic activity assay indicate that the constructs all containcatalytically active RNase moieties

Example 12 In Gel DNase Activity of RSLV Nuclease Molecules

FIG. 11 shows results from a DNase nuclease activity assay performed onprotein A purified protein from COS7 supernatants transfected with theRSLV fusion plasmids in Example 10. FIG. 11 shows fivee panels (11a,11b, 11c), with each gel panel showing the digestion pattern withdecreasing amounts of the indicated fusion protein and 1 microgram ofplasmid DNA. Each protein was serially diluted in two-fold increments innuclease free water from 500 ng to 4 ng of enzyme. To each sample, wasadded 1 ug PDG plasmid DNA, incubated for 30 minutes at 37 degrees.One-half of each sample was subjected to agarose gel electrophoresis for30 minutes at 100 volts using 1.2% TAE agarose gels. FIG. 11c shows theresits of an in gel DNase enzyme activity assay using commerciallyavailable DNase 1 (Biolabs, Inc.). The far right lane is a negativecontrol with the DNA alone and no enzyme, the lane to the left of thatis another negative control, this is an RNase-Ig molecule which hasRNase activity but no DNase activity, as expected the plasmid DNAremains intact and is not digested in both cases. The resultsdemonstrate that commercially available DNase1 enzyme is highly activeand digests all the DNA at most of the concentrations tested. Theresults in FIGS. 11a and 11b show the DNase activity of four differentnuclease Fc fusion constructs. The top panel in FIG. 11a shows theability of a DNase-Ig fusion protein to digest the plasmid DNA, as isapparent from the gel, this enzyme digests all the DNA at allconcentrations tested is as active, or more active than commerciallyavailable DNase 1. In the lower panel of FIG. 11a is a bi-specificnuclease Fc fusion protein with the DNase on the amino terminus of theFc (SED ID 65-66) and it also has robust DNase enzymatic activity, but alittle less than the DNase-Ig in the upper panel of FIG. 11 a. The toppanel of FIG. 11b shows the DNase enzymatic activity of anotherbi-specific nuclease, this Fc fusion protein has the DNase on theC-terminus of the Fc connected to the Fc via a specially engineered NLGlinker (SEQ ID 67-68). As is apparent by the data this enzyme also hasrobust DNase enzymatic activity and appears to be more active than theother bi-specific nucleases examined here. The bottom panel of FIG. 11bshows the DNase enzymatic activity of another bi-specific nucleasemolecule that is lacking the (G4S)4 linker connecting the RNase modulewith the Fc (SEQ ID 69-70). This bi-specific nuclease also has goodDNase activity but it appears to be somewhat less than the other twobi-specific nuclease Fc fusion proteins shown in this experiment. Thisdata suggests that all of the bi-specific nucleases have good DNaseactivity, which is an unexpected given the past efforts of others inthis regard (Dwyer et al. JBC; Vol 271, No. 14; pp 9738-9743).Furthermore the position of the DNase in the construct as well as thelinker length and composition connecting the DNase to the Fc arecritical in creating a highly active DNase enzyme in the context of abi-specific nuclease Fc fusion protein.

Example 13 Analysis of Enzyme Kinetics

FIGS. 12-13 show results from a kinetic fluorescence enzyme activityassay comparing the RNase enzyme activity of recombinant RNase A(Ambion), RSLV 125, RSLV 126, hRNase WT-SCCH-WThIgG1, andhRNaseG88D-SCCH-(P238S/K322S/P331S)hIgG1. To further define thefunctional characteristics of the bivalent mRNase-Ig fusion protein, westudied the enzyme kinetics of different nuclease fusion proteins usingthe RNase Alert Substrate (Ambion/IDT) and fluorescence was quantifiedwith a Biotek Synergy2 microplate reader. Data was analyzed using Gen5software (Biotek Instruments, Inc., Winooski, Vt.). Relativefluorescence units as a function of time were assayed very minute overthe course of a 45 minute experiment incubated at 37 C according tomanufacturer's instructions, using decreasing enzyme concentrationsstarting from 10 pg/ul and serially diluting to 0.1 pg/ul by 0.67×increments. Each sample included a fixed concentration of RNase Alertsubstrate (200 nM) in 1× RNase Alert Reaction Buffer.

FIG. 12 shows the RFU (relative fluorescence units) versus time for eachprotein at equimolar concentrations, with the test proteins at 4.5 pg/ulor 4.5 ng/ml, and the recombinant RNaseA control at 1.3 pg/ul in thepresence of 200 nM RNase Alert substrate.

FIG. 13 shows a Lineweaver Burk plot of the different molecules. Inorder to estimate the Vmax and Km, RNase Alert kinetic fluorescenceassays were set up using 105 pM enzyme, and the substrate concentrationwas decreased from 200 nM to 50 pM in four fold increments. Thus, theenzyme concentration was fixed and substrate concentration titrated inthis series of experiments. The data show the Lineweaver Burk plotsgenerated for the different fusion proteins under these conditions.Taken together the data in FIGS. 12 and 13 demonstrate that the RNasemoieties are highly active in the three RNase Fc fusion proteinsconstructed and tested here.

Example 14 Assessment of In Vitro Cytotoxicity Against Human THP-1 CellLine

FIGS. 14-15 show results of in vitro studies analyzing the effects ofRNase1g fusion proteins with wild type or mutant (including SCC, P238S,P331S) −IgG Fc domains on the survival of a human monocytic cell line,THP1. The THP1 cells were maintained in logarithmic growth in RPMI/10%FBS prior to harvest for the assays. Cells were greater than 98% viableprior to use in cytotoxicity assays. THP1 cells were plated in 96 wellplates at a cell density of 1×10e6 c/ml, or 100,000 cells per well.Hybrid nuclease proteins were added to successive wells using a two-foldserial dilution series starting at 5 micrograms/ml and ending with 0.01microgram/ml fusion protein per reaction. In this experiment an RNase-Fcfusion protein with a wild type IgG1 Fc (wtRNasewtIgG) was compared withan RNase-Fc with a mutant Fc (P238S, P331S) that had significantlyreduced Fc receptor binding and internalization (mtRNasemgIgG), withrespect to their ability to induce cytotoxicity in the cultured THP1cells. Reactions were incubated in 96 well plates for three days at 37°C., 5% CO2 prior to cell harvest and analysis. After three days, cellswere harvested by centrifugation at 1000 rpm, washed in PBS/2% FBS, andincubated with FITC Annexin V apoptosis detection kit reagents (#556547,Becton Dickinson/Pharmingen), according to manufacturer's instructions.Cells were washed with 100 microliters cold binding buffer supplied withthe kit, and annexin V-FITC/Propidium Iodide (PI) added at 1:100 in 100ul binding buffer. Samples were incubated on ice for 20 minutes, afterwhich 400 μl additional binding buffer was added to each sample. Stainedsamples were analyzed by flow cytometry using a FACS Canto (BectonDickinson) and data analyzed using Flowjo software (Treestar, Ashland,Oreg.).

FIG. 14 shows the effect of RNase Fc fusion proteins with a wild type ormutant Fc domain on cell death as measured by two methods, Annexin Vbinding (top panel) and propidium iodide binding (bottom panel), bothare sensitive measure of cell death. This experiment demonstrates thatbinding of the RNase fusion protein with mutant Fc (P238S, P331S) hasreduced binding to Fc recptors on the surface of the THP1 cells, andsubsequent internalization of the protein. The results show asignificant decrease in cell death by the RNase-Fc mutant compared toRNase-wild type Fc fusion proteins (e.g., an approximately 3-folddecrease at 1.25 μg/ml of protein). FIG. 15 presents the results offluorescence-activated cell sorting (FACS) experiments to examine thecytotoxicity of RNase Fc fusions constructs with a wild type or mutantFc domain (RNase-wtIgG or RNase-mtIgG respectively). The datademonstrate a significant decrease in the number of dead cells when theTHP1 cells are incubated with the RNase Fc construct with a mutant Fc(smaller peak size to the right of the graph for the RNase Fc mutantcompared to RNase-wtIgG). These data show an approximately 3- to 5-folddecrease in cell death by the RNase Fc mutant compared to wild type.These and other experiments examining Fc receptor binding clearly showthat in the presence of cells bearing Fc receptors the RNase Fcconstruct with a mutated Fc region has reduced binding to Fc receptorsand less internalization by cells, resulting in less cell death due tothe RNase activity of the construct. Such constructs are particularlyuseful in treating autoimmune diseases as it may be undesirable to use aprotein therapeutic which is cytotoxic to Fc receptor bearing cells.

Example 15 IFN-Alpha Production by Human PBMCs is Inhibited by RSLV-132Addition to Cultures In Vitro

RSLV132 addition abolished the induction of interferon-α from humanperipheral blood mononuclear cells stimulated using immune complexesformed with serum from three SLE patients plus necrotic cell extract(NCE) (FIG. 16). To measure the ability of RSLV-132 to bind to anddegrade RNA contained in the immune complexes of lupus patients, and invitro bioassay was developed. The experiment involves the formation ofimmune complexes in vitro using the autoantibodies from lupus patientsand NCE from cultured human cells (U937). Combining the lupus patientserum with the NCE results in the formation of immune complexes (IC)which are very potenti inducers of interferon, normal human serum doesnot stimulate the production of interferon. The IC are incubated withnormal human peripheral blood mononuclear cells (PBMC's) as reportercells. The production of interferon by the reporter cell sis measuredusing an interferon-α ELISA. Reporter cells were obtained from normalvolunteers by Ficoll density gradient centrifugation. Lupus patient orhealthy normal volunteer serum was obtained under University ofWashington Institutional Rreview Board #HSD No. 3971, the serum wasdiluted 1/1000 and added to 10% (v/v) of necrotic cell extract (NCE)derived from cultured U937 cells as above. Diluted lupus patient ornormal volunteer serum was incubated with the NCE for 15 minutes at roomtemperature, the resulting IC's were incubated with or without variousdoses of RSLV-132, RSLV-124, or wild type RNase for 15 minutes thenincubated with normal PBMC's for 20 hours in the presence of 500 U/mLUniversal IFN followed by measurement of the amount of IFN secreted fromthe PBMC culture. Serum was obtained from three (3) different lupuspatients with various degrees of disease activity ranging from mild toactive. The NCE was incubated with either lupus patient serum or healthynormal volunteer serum at room temperature for 15 minutes followed by 20hour incubation with PBMC's. IFN-α was quantitated by ELISA where IFN-αis captured using a mouse MAb against human IFN alpha (MMHA-11) [PBLBiomedical Laboratories, product #2112-1] and was detected using arabbit polyclonal antibody against IFN alpha [PBL BiomedicalLaboratories, product #31101-1], followed by development usinganti-rabbit HRP [Jackson Immuno Research, product #711-035-152] and TMBsubstrate. In some cases prior to addition of the NCE to the PBMC's, thetest article (RSLV-124 or RSLV-132) was added at concentrations of 0.16,0.5, 1.6 and 5.0 ug/mL or RNAse was added at concentrations of 0.05,0.16, 0.5 and 1.6 ug/mL (equimolar) to the NCE. The ability of the lupuspatient serum to stimulate production of IFN from the PBMC's was reducedby approximately 50% with the addition of 5.0 ug/mL RSLV-124. Thisinhibition mirrored that of an equimolar quantity of RNAse. Addition ofthe same concentration of huRSLV-132 was as or more effective atinhibiting IFN than RSLV-124, with almost completely abolished IFNproduction with the addition of 5.0 ug/mL of huRSLV-132. When combinedwith NCE, lupus patient anti-RNA/DNA antibodies are potent induces ofIFN from freshly isolated PBMC's. Serum from normal volunteers does nothave this same ability to stimulate IFN production from the reportercells. This data indicates that the auto-antibodies circulating in lupuspatient serum are able to form immune complexes which presumably triggerTLR7 and the subsequent production of IFN. The exact type and subtypesof IFN were not analyzed. This data indicates that RSLV-132 binds to itsmolecular target, the RNA associated with the lupus patient IC's andpotently degrades it, thereby preventing the stimulation of IFN from thePBMC's (FIG. 16). RSLV-132 appears to be more active than RSLV-124 inthis assay.

Example 16 RSLV-132 is a Potent In Vivo Inhibitor of RNA-InducedInterferon Activation

To assess the ability of RSLV-132 to bind to and degrade RNA in thecirculation of the mouse, a pharmacodynamic model was developed using anRNA mimetic polyinosinic:polycytidilic acid (poly I:C) which is a robustactivator of the interferon pathway. Poly(I:C) is a mismatcheddouble-stranded RNA with one strand being a polymer of inosinic acid,the other a polymer of cytidylic acid. It is know to interact withtoll-like receptor 3 (TLR3) which is expressed in the membrane of Bcells, macrophages and dendritic cells. Poly(I:C) is available fromInvitrogen. The effects of poly I:C can be quantitated by measuring theexpression levels of interferon stimulated genes in the spleen of themouse following administration. On day zero 10 B6 mice, three months ofage, were treated with either RSLV-132 (250 ug per mouse) or intravenousimmunoglobulin (IVIG) (Privigen, Behring) (250 ug per mouse) as acontrol, both via intraperitoneal injection. Twenty hours afterinjection of RSLV-132 or IVIG, poly(I:C) was injected into the mice at200 ug per mouse intraperitoneally. Two hours later, the animals weresacrificed by CO2 exposure, the spleens were collected into RNAlater(Qiagen) and stored at −80 C for later study of the expression ofInterferon stimulated genes (ISGs). Spleen samples were subjected tostudy of the expression of ISGs, including Ifitl (Interferon-inducedprotein with tetratricopeptide repeats 1 (UniProt Q64282)), Irf7(Interferon regulatory factor 7 (UniProt P70434)) and Mx1 gene by qPCR.The results from these experiments demonstrate that intraperitonealinjection of RSLV-132 results in serum concentrations of RSLV-132 whichare able to bind to circulating poly (I:C) and effectively degrade theRNA mimetic, thereby effectively preventing stimulation of theInterferon pathway and the three ISG's monitored (FIG. 17).

Example 17 Analysis of Enzyme Kinetics for RSLV-132 and RSLV-133

RSLV-132 and RSLV-133 were transiently expressed in CHO cells andpurified using protein-A. The RNase activity of these RNase Fc fusionproteins was quantitated using the RNaseAlert QC kit from Ambion (Cat#AM1966). Various amounts of the RNase Fc fusion protein were used andthe results shown in FIG. 18 in relative fluorescence units (RFU's) overtime. The results demonstrate that RSLV-132 is a highly active RNaseenzyme, and has increased RNase activity relative to other RNase Fcfusion constructs such as RSLV-124 and wild type RNase For example usingequal amounts (400 pM) of RSLV132 and RSLV-124 yields more than twicethe RFU's (80,000 vs. 35,000) for RSLV-132 vs. RSLV-124. In addition,two production lots were tested for their stability at 4 C. RSLV-132.1was stored at 4 C for 8 weeks prior to this experiment and RSLV-132.2was stored at −80 C and thawed just prior to testing, demonstrating thatthe protein is stable at 4 C for up to 2 months. The stability of thedrug and increased catalytic activity may provide increased efficacy ina therapeutic setting.

FIG. 19 shows the RNase enzymatic activity in RFUs over time, comparingthe amount of RNase activity of the bi-specific RSLV-133 molecule withthe monospecific RSLV-132, and wild type RNase. As demonstrated in FIG.19 the RSLV-133 molecule has significantly increased RNase activityrelative to the monospecific RSLV-124 molecule or, an earlier bispecificnuclease Fc, RSLV-123, or wild type RNase, yielding greater than 2-foldmore RFU's with an equal amount of protein. FIG. 20 show the results ofa DNase enzymatic activity assay of the RLSV-133 molecule in comparisonto RSLV-123, a previous bi-specific nuclease construct, and wild typeDNase. In this experiment DNase enzymatic activity was quantitated usingthe DNaseAlert Kit from Integrated DNA Technologies, Cleavage of the DNAsubstrate yielded a fluorescent emission which was quantitated using aSynergy2 Multi-Mode Microplate Reader (BioTek Instruments, Inc.,Winooski, Vt.). FIG. 20 shows the RFU's of DNase enzymatic activity overtime for RSLV-133, RSLV-123, or wild type DNase. The results of theexperiment demonstrate that RSLV-133 has increased DNase activityrelative to wild type DNase and RSLV-123, the earlier bi-specificnuclease molecule, yielding more than 3-fold more DNase activity in thelinear range of the experiment. FIG. 21 demonstrates the ability of theRSLV-133 molecule to digest DNA in an in-gel digestion experiment. Theresults show that RSLV-133 is able to digest DNA in this assay aseffectively as wild type DNase (compare lanes 5&7). Given the relativemolecular weights of RSLV-133 and wild type DNase it appears thatRSLV-133 is more effective in digesting DNA in this assay as well.

Example 18 RSLV-132 Demonstrates decreased Fc Receptor Binding

To examine the ability of RNase Fc fusion proteins to bind Fc receptorsin vitro, RSLV124 (wild type Fc domain) and RSLV-132 (mutant Fc domain;P238S/P331S) were incubated with an Fc bearing human myeloid cell line,THP1 and the specific binding to the cells was quantitated byfluorescence-activated cell sorting (FACS) analysis. RLSV-124 andRSLV-132 were fluorescently labeled using alexa fluor dye AF-647 fromInvitrogen (Cat #A20006). After dialyzing the RNase Fc fusion proteinsto remove the unbound dye, varying amounts of the labeled proteins wereincubated with THP1 cells for one hour, the cells were washedstringently to remove unbound RNase Fc fusion protein, and thespecifically bound protein was quantitated by FACS measuring meanfluorescence intensity. The results in FIG. 22 demonstrate that theRSLV-132 protein which has a mutant Fc domain has significantly less Fcreceptor binding than RSLV-124 which has a wild type Fc domain,exhibiting greater than 4-fold reduction in Fc receptor binding Thisfinding is consistent with our previous findings that RNase Fc fusionproteins with a mutant Fc domain (P238S/P331S) have significantlydecreased cytotoxicity.

Example 19 In Vitro Assessment of Hybrid Nuclease Molecule BiologicalActivity

One or more hybrid nuclease molecules are purified, e.g., by affinity orion exchange chromatography as previously described in the examplesabove. In some instances the hybrid nuclease molecule is a polypeptide.In some instances, the hybrid nuclease molecule includes one or moresequences from Table 1. In some instances, the hybrid nuclease moleculeincludes a nuclease domain linked to a mutant Fc domain. In someinstances, the hybrid nuclease molecule includes a mutant Fc domain. Insome instances, the mutant Fc domain comprises mutations in the hinge,CH2, and/or CH3 domains. In some instances, the mutant Fc domaincomprises P238S and/or P331S, and may include mutations in one or moreof the three hinge cysteines. In some instances, the mutant Fc domaincomprises P238S and/or P331S, and/or mutations in the three hingecysteines. In some instances, the mutant Fc domain comprises P238Sand/or P331S, and/or mutations in the three hinge cysteines to SSS. Insome instances, the mutant Fc domain comprises P238S and P331S andmutations in the three hinge cysteines. In some instances, the mutant Fcdomain comprises P238S and P331S and SSS. In some instances the mutantFc domain is shown in SEQ ID NOs 59, 60, 61. In some instances, thehybrid nuclease molecule is shown in SEQ ID NOs. Various linker domains(e.g., those described herein) can be used to link the Fc domains tonuclease domains. For example, linker domains 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acidsin length can be used. Molecules are assayed for the specific nucleaseactivity in vitro using qualitative assays to verify that they possessthe desired nuclease function. Specific activities are generally thendetermined by fluorescence based kinetic assays utilizing substratessuch as the RNase or DNase Alert Kit reagents, and a fluorescence platereader set to take readings as a function of time. In addition, proteinsolutions are generally checked for endotoxin contamination using acommercially available kits, such as the Pyrotell Limulus AmebocyteLysate (LAL) kit, 0.06 EU/ml detection limit from Cape Cod, Inc. (E.Palmouth, Mass.). Molecules are then assayed using a variety of in vitroassays for biological activity.

One series of in vitro assays will measure the effect of the moleculeson cytokine production by human PBMC in response to various stimuli, inthe presence or absence of the molecules in the cultures. Normal orpatient human PBMC (approximately 1×10e6 cells) are cultured for 24, 48,or 96 hours depending on the assay. PBMC are cultured in the presence ofstimuli such as TLR ligands, costimulatory antibodies, immune complexes,and normal or autoimmune sera. The effects of the molecules on cytokineproduction is measured using commercially available reagents, such asthe antibody pair kits from Biolegend (San Diego, Calif.) for IL-6,IL-8, IL-10, IL-4, IFN-gamma, TNF-alpha. Culture supernatants from invitro cultures are harvested at 24, 48 hours or later time points todetermine the effects of the molcules on cytokine production. IFN-alphaproduction is measured using, e.g., anti-human IFN-alpha antibodies andstandard curve reagents available from PBL interferon source(Piscataway, N.J.). A similar set of assays is performed using humanlymphocyte subpopulations (isolated monocytes, B cells, pDCs, T cells,etc.); purified using, e.g., commercially available magnetic bead basedisolation kits available from Miltenyi Biotech (Auburn, Calif.).

In addition, the effect of the molecules on expression of lymphocyteactivation receptors such as CDS, CD23, CD69, CD80, CD86, and CD25 isassessed at various time points after stimulation. PBMC or isolated cellsubpopulations are subjected to multi-color flow cytometry to determinehow these molecules affect the expression of different receptorsassociated with immune cell activation.

Another set of assays will measure the effects of these molecules on theproliferation of different lymphocyte subpopulations in vitro. Theseassays will utilize, e.g., CI-DA-SE staining (Invitrogen, Carlsbad, CA)of human PBMCs prior to stimulation. CFSE at 5 mM is diluted 1:3000 inPBS/0.5% BSA with 10e7-10e8 PBMCS or purified cell subsets and labelingreactions incubated for 3-4 minutes at 37 C prior to washing severaltimes in RPMI/10% FBS to remove remaining CFSE. CFSE labeled cells arethen incubated in co-culture reactions with various stimuli (TLRligands, costimulatory antibodies, etc.) and the molecules for 4 daysprior to analysis of cell proliferation by flow cytometry usingdye-conjugated cell subpopulation specific antibodies.

Another assay will measure the cytotoxicity of one or more molecules.This assay will measure toxicity using Annexin 5 staining (e.g., Annexin5-FITC). Cells of interest (e.g., monocytes or a monocyte cell line) arecontacted with a hybrid nuclease molecule of interest (e.g., a hybridnuclease molecule having a mutant Fc domain) or one or more controls. Atvarious time points following contact, cells are separated from cultureand stained with Annexin 5. The number of apoptotic or dead cells arethen counted, e.g., using flow cytometry or a fluorescence microscope.Cells contacted with a hybrid nuclease molecule of interest show lowernumbers of cells staining positive for Annexin 5 compared to positivecontrols.

The effect of these molecules on in vitro maturation of monocytes intoDCs and macrophages is also assessed using both normal and patient PBMCsamples.

The effectiveness of a hybrid nuclease molecule is demonstrated bycomparing the results of an assay from cells treated with a hybridnuclease molecule disclosed herein to the results of the assay fromcells treated with control formulations. After treatment, the levels ofthe various markers (e.g., cytokines, cell-surface receptors,proliferation) described above are generally improved in an effectivemolecule-treated group relative to the marker levels existing prior tothe treatment, or relative to the levels measured in a control group.

Example 20 Administration of a Hybrid Nuclease Molecule to a Mammal inNeed Thereof

Mammals (e.g., mice, rats, rodents, humans, guinea pigs) are used in thestudy. Mammals are administered (e.g., intravenously) one or more hybridnuclease molecules comprising one or more sequences from Table 1 or acontrol. In some instances the hybrid nuclease molecule is apolypeptide. In some instances, the hybrid nuclease molecule includesone or more sequences from Table 1. In some instances, the hybridnuclease molecule includes a nuclease domain linked to a mutant Fcdomain. In some instances, the hybrid nuclease molecule includes amutant Fc domain. In some instances, the mutant Fc domain comprisesmutations in the hinge, CH2, and/or CH3 domains. In some instances, themutant Fc domain comprises P238S and/or P331S, and may include mutationsin one or more of the three hinge cysteines. In some instances, themutant Fc domain comprises P238S and/or P331S, and/or mutations in thethree hinge cysteines. In some instances, the mutant Fc domain comprisesP238S and/or P331S, and/or mutations in the three hinge cysteines toSSS. In some instances, the mutant Fc domain comprises P238S and P331Sand mutations in the three hinge cysteines. In some instances, themutant Fc domain comprises P238S and P331S and SSS. In some instancesthe mutant Fc domain is shown in SEQ ID NOs 59, 60, 61. In someinstances, the hybrid nuclease molecule is shown in SEQ ID NOs. Variouslinker domains (e.g., those described herein) can be used to link the Fcdomains to nuclease domains. For example, linker domains 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or moreamino acids in length can be used. In some instances the hybrid nucleasemolecule is formulated a pharmaceutically acceptable carrier. In someinstances the molecule is formulated as described in the pharmaceuticalcompositions section above. The hybrid nuclease molecule targets RNaseand/or DNase.

Multiple rounds of doses are used where deemed useful. Effects onIFN-alpha levels, IFN-alpha response gene levels, autoantibody titers,kidney function and pathology, and/or circulating immune complex levelsare monitored in the mammals. Similar studies are performed withdifferent treatment protocols and administration routes (e.g.,intramuscular administration, etc.). The effectiveness of a hybridnuclease molecule is demonstrated by comparing the IFN-alpha levels,IFN-alpha response gene levels, autoantibody titers, kidney function andpathology, and/or circulating immune complex levels in mammals treatedwith a hybrid nuclease molecule disclosed herein to mammals treated withcontrol formulations.

In an example, a human subject in need of treatment is selected oridentified. The subject can be in need of, e.g., reducing a cause orsymptom of SLE. The identification of the subject can occur in aclinical setting, or elsewhere, e.g., in the subject's home through thesubject's own use of a self-testing kit.

At time zero, a suitable first dose of a hybrid nuclease molecule isadministered to the subject. The hybrid nuclease molecule is formulatedas described herein. After a period of time following the first dose,e.g., 7 days, 14 days, and 21 days, the subject's condition isevaluated, e.g., by measuring IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels. Other relevant criteria can also bemeasured. The number and strength of doses are adjusted according to thesubject's needs.

After treatment, the subject's IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels are lowered and/or improved relativeto the levels existing prior to the treatment, or relative to the levelsmeasured in a similarly afflicted but untreated/control subject.

In another example, a rodent subject in need of treatment is selected oridentified. The identification of the subject can occur in a laboratorysetting or elsewhere.

At time zero, a suitable first dose of a hybrid nuclease molecule isadministered to the subject. The hybrid nuclease molecule is formulatedas described herein. After a period of time following the first dose,e.g., 7 days, 14 days, and 21 days, the subject's condition isevaluated, e.g., by measuring IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels. Other relevant criteria can also bemeasured. The number and strength of doses are adjusted according to thesubject's needs.

After treatment, the subject's IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels are lowered and/or improved relativeto the levels existing prior to the treatment, or relative to the levelsmeasured in a similarly afflicted but untreated/control subject.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

TABLE 1 SEQ ID NO: DESCRIPTION SEQUENCE (NUCLEOTIDE SEQUENCES ARE 5′-3′)30 g4s4lnk agatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgag 31 G4S5-1agatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtaccggtctcgag 32 G4S5-2agatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtctcgag 33 3′hRNaseG88Dgtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacgactccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 34 hDNase1-3′-gatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatg G105R; A114Ftccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgataatctaga 35 hDNase1-3′-gatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatg WTtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaatgataatctaga 36 hDNase1-gatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatg 3′A114Ftccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttagaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgataatctaga 37 hDNase1-5′-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg G105R; A114Ftccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgag 38 hDNase1-5′-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg WTtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgag 39 hDNase1-5′-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg A114Ftccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttagaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgag 40 hIgG1(SCC)agatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaa atgataatctaga 41hDNase1 + VK3 gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc LPtggctcccagataccaccggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtga 42 hDNase1L3atgtcacgggagctggccccactgctgcttctcctcctctccatccacagcgccctggccatgaggatctgctccttcaacgtcaggtcctttggggaaagcaagcaggaagacaagaatgccatggatgtcattgtgaaggtcatcaaacgctgtgacatcatactcgtgatggaaatcaaggacagcaacaacaggatctgccccatactgatggagaagctgaacagaaattcaaggagaggcataacatacaactatgtgattagctctcggcttggaagaaacacatataaagaacaatatgcctttctctacaaggaaaagctggtgtctgtgaagaggagttatcactaccatgactatcaggatggagacgcagatgtgttttccagggagccctttgtggtctggttccaatctccccacactgctgtcaaagacttcgtgattatccccctgcacaccaccccagagacatccgttaaggagatcgatgagttggttgaggtctacacggacgtgaaacaccgctggaaggcggagaatttcattttcatgggtgacttcaatgccggctgcagctacgtccccaagaaggcctggaagaacatccgcttgaggactgaccccaggtttgtttggctgatcggggaccaagaggacaccacggtgaagaagagcaccaactgtgcatatgacaggattgtgcttagaggacaagaaatcgtcagttctgttgttcccaagtcaaacagtgtttttgacttccagaaagcttacaagctgactgaagaggaggccctggatgtcagcgaccactttccagttgaatttaaactacagtcttcaagggccttcaccaacagcaaaaaatctgtcactctaaggaagaaaacaaagagcaaacgctcctag 43 humanatgggtctggagaagtctcttgtccggctccttctgcttgtcctgatactgctg pancreaticgtgctgggctgggtccagccttccctgggcaaggaatcccgggccaagaaattc ribonucleasecagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgctactgtgtag 44 NLG linkergtcgacggcgcggccgccagccccgtgaacgtgagcagccccagcgtgcaggat atc 45 g4s4lnkggggsggggsggggsggggs 46 G4S5-1 ggggsggggsggggsggggsggggs 47 G4S5-2ggggsggggsggggsggggsggggs 48 hDNase1-3′-lkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkildn G105R;A114Flnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk* 49 hDNase1-3-lkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkildn WTlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk* 50 hDNase1-lkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkildn 3′A114Flnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk* 51 hDNase1-5′-lkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkildn G105Rlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk 52 hDNase1-5′-lkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkildn WTlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk 53 hDNase1-5′-lkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkildn A114Flnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk 54 hIgG1(SCC)lepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhe alhnhytqkslslspgk55 hRNase- kesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvG88D-3′ qnvcfqekvtckngqgncyksnssmhitdcrltndsrypncayrtspkerhiivacegspyvpvhfdasvedst* 56 humanmetpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi DNase1 + VK3LPalvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqais dhypvevmlk* 57DNase1L3 msrelapllllllsihsalamricsfnvrsfgeskqedknamdvivkvikrcdiilvmeikdsnnricpilmeklnrnsrrgitynyvissrlgrntykeqyaflykeklvsvkrsyhyhdyqdgdadvfsrepfvvwfqsphtavkdfviiplhttpetsvkeidelvevytdvkhrwkaenfifmgdfnagcsyvpkkawknirlrtdprfvwligdqedttvkkstncaydrivlrgqeivssvvpksnsvfdfqkayklteeealdvsdhfpvefklqssraftnskksvtlrkktkskrs* 58 humanmalekslvrllllvlillvlgwvqpslgkesrakkfqrqhmdsdsspsssstyc pancreaticnqmmrrrnmtqgrckpvntfvheplvdvqnvcfclekvtckngqgncyksnssmh ribonucleaseitdcrltngsrypncayrtspkerhiiyacegspyypyhfdasyedst (Uniprot P07998) 59Fc domain cccaaatcttctgacaaaactcacacatctccaccgtctccagcacctgaactcwith SSS ctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaa 60 Fc domainpkssdkthtsppspapellggpsyflfppkpkdtlmisrtpevtcvvvdvshed with SSSpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheal hnhytqkslslspgk61 RSLV125: atggaaacccctgcccagctgctgttcctgctgctgctgtggctgcccgacacchuVK3LP- accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagacwthRNase- agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatSSS-mthIgG1 atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtaP238S P331S gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctaccctcgagcccaaatcttctgacaaaactcacacatctccaccgagcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctct ccgggaaaatga 62RSLV125: metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn wthRNase-gsrypncayrtspkerhiivacegspyvpvhfdasvedstlepkssdkthtspp SSS-mthIgG1spapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv P238S P331Sevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsls pgk 63 RSLV126:atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagatacc huVK3LP-accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagac WThRNase-agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaat (g4s)4-SSS-atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggta mthIgG1-gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggc P2385-P3315aactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatctccaccgagcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaatga 64 RSLV126:metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn WThRNase-gsrypncayrtspkerhiivacegspyvpvhfdasvedstggggsggggsgggg (g4s)4-SSS-sggggslepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcv mthIgG1-vvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng P2385-P3315keykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 65 RSLV127:atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagatacc huVK3LP-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg hDNase1tccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatc 105/114-gccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctg (g4s)4-SSS-gacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagcca mthIgG1-ctgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccag P238S-gtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaac P3315-NLG-gacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagag RNasegtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggttctaccggtctcgagcccaaatcttctgacaaaactcacacatctccaccgagcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaagtcgacggagctagcagccccgtgaacgtgagcagccccagaatgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaagtggtacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccgaaccagcccgaaggagagacacatcattgtggcctgtgaaggagcccatatgtgccagtccactttgatgcttgctgtggag gactctacctaa 66RSLV127: metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi huVK3LP-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq hDNase1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 105/114-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (g4s)4-SSS-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqais mthIgG1-dhypvevmlkggggsggggsggggsggggslepkssdkthtsppspapellggs P238S-svflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpr P3315-NLG-eeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprep RNaseqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdikesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst 67 RSLV128:atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagatacc huVK3LP-accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagac hRNase WT-agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaat (g4s)4-SSS-atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggta mthIgG1-gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggc P238S-aactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaac P3315-NLG-ggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatc hDNaseattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtg 105/114gaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatctccaccgagcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaagtcgacggagctagcagccccgtgaacgtgagcagccccagaatgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaa tga 68 RSLV128:metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNase WT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstggggsggggsgggg (g4s)4-SSS-sggggslepkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcv mthIgG1-vvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng P238S-keykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvk P3315-NLG-gfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfs hDNasecsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdilkiaafniqtfge 105/114tkmsnatlvsyivqilsrydialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk 69 RSLV129:atggaaacccctgcccagctgctgttcctgctgctgctgtggctgcccgacacc huVK3LP-accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagac hRNAseWT-agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaat SSS-atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggta mthIgG1-gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggc P238S-aactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaac P3315-NLG-ggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatc hDNAseattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtg 105/114gaggactctaccctcgagcccaaatcttctgacaaaactcacacatctccaccgagcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaagtcgacggagctagcagccccgtgaacgtgagcagccccagaatgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaatga 70 RSLV129:metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNAseWT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstlepkssdkthtspp SSS-spapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv mthIgG1-evhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiekti P238S-skakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpenn P3315-NLG-ykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsls hDNAsepgkvdgasspvnvsspsvqdilkiaafniqtfgetkmsnatlvsyivqilsryd 105/114ialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqai sdhypvevmlk 71Fc domain cccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcwith P238S- ctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatg 2atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagac (SCC hinge)cctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccctatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaa 72 Fc domainpkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtavvvdvshed with P238S-pevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvs 2nkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdia (SCC hinge)vewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheal hnhytqkslslspgk73 Fc domain cccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcwith P331S- ctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg 2atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaa 74 Fc domainpkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshed with P331S-pevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvs 2nkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheal hnhytqkslslspgk75 Fc domain cccaaatcttctgacaaaactcacacatctccaccgagcccagcacctgaactcwith SSS, ctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgP238S, and atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagacP331S-2 cctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaa 76 Fc domainpkssdkthtsppspapellggssvflfppkpkdtlmisrtpevtcvvvdvshed with SSS,pevkfnwyydgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvs P238S, andnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdia P331S-2vewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheal hnhytqkslslspgk77 RSLV125-2: atggaaacccctgcccagctgctgttcctgctgctgctgtggctgcccgacacchuVK3LP- accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagacwthRNase- agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatSCC-mthIgG1 atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtaP238S P331S gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctaccctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctct ccgggaaaatga 78RSLV125-2: metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrnhuVK3LP- mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltnwthRNase- gsrypncayrtspkerhiivacegspyvpvhfdasvedstlepkssdkthtcppSCC-mthI gG1 cpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvP238S P331S evhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsls pgk 79 RSLV126-2:atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagatacc huVK3LP-accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagac WThRNase-agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaat (g4s)4-SCC-atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggta mthIgG1-gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggc P2385-P3315aactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaatga 80 RSLV126-2:metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn WThRNase-gsrypncayrtspkerhiivacegspyvpvhfdasvedstggggsggggsgggg (g4s)4-SCC-sggggslepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcv mthIgG1-vvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng P2385-P3315keykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 81 RSLV127-2:atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagatacc huVK3LP-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg hDNase1tccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatc 105/114-gccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctg (g4s)4-SCC-gacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagcca mthIgG1-ctgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccag P238S-gtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaac P3315-NLG-gacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagag RNasegtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaagtcgacggagctagcagccccgtgaacgtgagcagccccagaatgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaagtggtacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccgaaccagcccgaaggagagacacatcattgtggcctgtgaaggagcccatatgtgccagtccactttgatgcttgctgtggag gactctacctaa 82RSLV127-2: metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydihuVK3LP- alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq hDNase1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 105/114-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (g4s)4-SCC-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqais mthIgG1-dhypvevmlkggggsggggsggggsggggslepkssdkthtcppcpapellggs P238S-svflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpr P3315-NLG-eeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprep RNaseqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdikesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst 83 RSLV128-2:atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagatacc huVK3LP-accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagac hRNase WT-agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaat (g4s)4-SCC-atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggta mthIgG1-gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggc P238S-aactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaac P3315-NLG-ggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatc hDNaseattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtg 105/114gaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaagtcgacggagctagcagccccgtgaacgtgagcagccccagaatgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaa tga 84 RSLV128-2:metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNase WT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstggggsggggsgggg 4s)4-SCC-sggggslepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcv mthIgG1-vvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng P238S-keykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvk P3315-NLG-gfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfs hDNasecsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdilkiaafniqtfge 105/114tkmsnatlvsyivqilsrydialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk 85 RSLV129-2:atggaaacccctgcccagctgctgttcctgctgctgctgtggctgcccgacacc huVK3LP-accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagac hRNAseWT-agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaat SCC-atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggta mthIgG1-gatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggc P238S-aactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaac P3315-NLG-ggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatc hDNAseattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtg 105/114gaggactctaccctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaagtcgacggagctagcagccccgtgaacgtgagcagccccagaatgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaatga 86 RSLV129-2:metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNAseWT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstlepkssdkthtcpp SCC-cpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv mthIgG1-evhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiekti P238S-skakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpenn P3315-NLG-ykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsls hDNAsepgkvdgasspvnvsspsvqdilkiaafniqtfgetkmsnatlvsyivqilsryd 105/114ialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqai sdhypvevmlk 87Fc domain cccaaatcttctgacaaaactcacacatgtccaccgtgtccagcacctgaactcwith SCC ctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaa 88 Fc domainpkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshed with SCCpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheal hnhytqkslslspgk89 Fc domain cccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcwith SCC, ctgggaggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgP238S, and atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagacP331S-2 cctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctctctccgggaaaa 90 Fc domainpkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshed with SCC,pevkfnwyydgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvs P238S, andnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdia P331S-2vewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheal hnhytqkslslspgk91 RSLV132: atggaaacccctgcccagctgctgttcctgctgctgctgtggctgcctgacacchuVK3LP- accggcaaagagtcccgggccaagaagttccagcggcagcacatggactccgacwthRNase- tccagcccttccagctcctccacctactgcaaccagatgatgcggcggagaaacSCC-mthIgG1 atgacccagggccggtgcaagcccgtgaacacctttgtgcacgagcccctggtgP238S P331S gacgtgcagaacgtgtgttttcaagagaaagtgacctgcaagaacggccagggcaactgctacaagtccaactcctccatgcacatcaccgactgccggctgaccaacggctccagataccccaactgcgcctaccggacctcccccaaagaacggcacatcatcgtggcctgcgagggctctccttacgtgcccgtgcacttcgacgcctccgtggaagattccaccctggaacccaagtcctccgacaagacccacacctgtcccccttgtcctgcccctgaactgctgggcggctcctccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacatgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggggtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggaaaagagtacaagtgcaaggtgtccaacaaggccctgcccgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagcagggacgagctgaccaagaaccaggtgtccctgacctgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacccagaagtccctgtccctgagc cccggcaaatga 92RSLV132: metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn wthRNase-gsrypncayrtspkerhiivacegspyypvhfdasvedstlepkssdkthtcpp SCC-mthIgG1cpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv P238S P331Sevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsls pgk 93 RSLV133:atggaaacccctgcccagctgctgttcctgctgctgctgtggctgcccgacacc huVK3LP-accggcaaagagagccgggccaagaagttccagcggcagcacatggacagcgac hRNAseWT-agcagccccagcagctccagcacctactgcaaccagatgatgcggcggagaaac SCC-atgacccagggccggtgcaagcccgtgaacaccttcgtgcacgagcccctggtg mthIgG1-gacgtgcagaacgtgtgttttcaagaaaaagtgacctgcaagaacggccagggc P238S-aactgctacaagagcaacagcagcatgcacatcaccgactgccggctgaccaac P3315-NLG-ggcagcagataccccaactgcgcctaccggaccagccccaaagaacggcacatc hDNAseatcgtggcctgcgagggcagcccttacgtgcccgtgcactttgacgccagcgtg 105/114gaagatagcaccctggaacccaagagcagcgacaagacccacacctgtcccccctgccctgcccctgagctgctgggcggaagcagcgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtctccaacaaggccctgcccgccagcatcgagaaaaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacacactgccccccagccgggacgagctgaccaagaaccaggtgtccctgacctgcctggtgaaaggcttctaccccagcgatatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtacagcaagctgaccgtggacaagagccggtggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggcaaggtggacggcgccagctcccctgtgaacgtgtccagccccagcgtgcaggacatcctgaagatcgccgccttcaacatccagaccttcggcgagacaaagatgagcaacgccaccctggtgtcctacatcgtgcagatcctgagcagatacgatatcgccctggtgcaagaagtgcgggacagccacctgaccgccgtgggcaagctgctggacaacctgaaccaggacgcccccgacacctaccactacgtggtgtccgagcctctgggccggaacagctacaaagaaagatacctgttcgtgtaccggcccgatcaggtgtccgccgtggacagctactactacgacgacggctgcgagccctgccggaacgacaccttcaaccgcgagcccttcatcgtgcggttcttcagccggttcaccgaagtgcgcgagttcgccatcgtgcccctgcatgctgcccctggcgacgccgtggccgagatcgatgccctgtacgacgtgtacctggatgtgcaagaaaagtggggcctggaagatgtgatgctgatgggcgacttcaacgccggctgcagctacgtgcggcccagccagtggtccagcatcagactgtggacctcccccaccttccagtggctgatccccgacagcgccgataccaccgccacccccacccactgtgcctacgacagaatcgtggtggccggcatgctgctgagaggcgccgtggtgcctgacagcgccctgccattcaattttcaagccgcctacggcctgagcgatcagctggcccaggccatcagcgaccactaccccgtggaagtgatgctgaagtga 94 RSLV133:metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn huVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNAseWT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstlepkssdkthtcpp SCC-cpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv mthIgG1-evhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiekti P238S-skakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpenn P3315-NLG-ykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsls hDNAsepgkvdgasspvnvsspsvqdilkiaafniqtfgetkmsnatlvsyivqilsryd 105/114ialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdgvsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqai sdhypvevmlk 95RSLV132: aaagagtcccgggccaagaagttccagcggcagcacatggactccgactccagcwthRNase- ccttccagctcctccacctactgcaaccagatgatgcggcggagaaacatgaccSCC-mthIgG1 cagggccggtgcaagcccgtgaacacctttgtgcacgagcccctggtggacgtgP238S P331S cagaacgtgtgttttcaagagaaagtgacctgcaagaacggccagggcaactgctacaagtccaactcctccatgcacatcaccgactgccggctgaccaacggctccagataccccaactgcgcctaccggacctcccccaaagaacggcacatcatcgtggcctgcgagggctctccttacgtgcccgtgcacttcgacgcctccgtggaagattccaccctggaacccaagtcctccgacaagacccacacctgtcccccttgtcctgcccctgaactgctgggcggctcctccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacatgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattggtacgtggacggggtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggaaaagagtacaagtgcaaggtgtccaacaaggccctgcccgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagcagggacgagctgaccaagaaccaggtgtccctgacctgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacccagaagtccctgtccctgagccccggc aaatga 96RSLV132: kesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvwthRNase- qnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivSCC-mthIgG1 acegspympvhfdasvedstlepkssdkthtcppcpapellggssvflfppkpkP238S P331S dtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 97 RSLV133:aaagagagccgggccaagaagttccagcggcagcacatggacagcgacagcagc hRNAseWT-cccagcagctccagcacctactgcaaccagatgatgcggcggagaaacatgacc SCC-cagggccggtgcaagcccgtgaacaccttcgtgcacgagcccctggtggacgtg mthIgG1-cagaacgtgtgttttcaagaaaaagtgacctgcaagaacggccagggcaactgc P238S-tacaagagcaacagcagcatgcacatcaccgactgccggctgaccaacggcagc P3315-NLG-agataccccaactgcgcctaccggaccagccccaaagaacggcacatcatcgtg hDNAsegcctgcgagggcagcccttacgtgcccgtgcactttgacgccagcgtggaagat 105/114agcaccctggaacccaagagcagcgacaagacccacacctgtcccccctgccctgcccctgagctgctgggcggaagcagcgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaacagcacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtctccaacaaggccctgcccgccagcatcgagaaaaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacacactgccccccagccgggacgagctgaccaagaaccaggtgtccctgacctgcctggtgaaaggcttctaccccagcgatatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtacagcaagctgaccgtggacaagagccggtggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggcaaggtggacggcgccagctcccctgtgaacgtgtccagccccagcgtgcaggacatcctgaagatcgccgccttcaacatccagaccttcggcgagacaaagatgagcaacgccaccctggtgtcctacatcgtgcagatcctgagcagatacgatatcgccctggtgcaagaagtgcgggacagccacctgaccgccgtgggcaagctgctggacaacctgaaccaggacgcccccgacacctaccactacgtggtgtccgagcctctgggccggaacagctacaaagaaagatacctgttcgtgtaccggcccgatcaggtgtccgccgtggacagctactactacgacgacggctgcgagccctgccggaacgacaccttcaaccgcgagcccttcatcgtgcggttcttcagccggttcaccgaagtgcgcgagttcgccatcgtgcccctgcatgctgcccctggcgacgccgtggccgagatcgatgccctgtacgacgtgtacctggatgtgcaagaaaagtggggcctggaagatgtgatgctgatgggcgacttcaacgccggctgcagctacgtgcggcccagccagtggtccagcatcagactgtggacctcccccaccttccagtggctgatccccgacagcgccgataccaccgccacccccacccactgtgcctacgacagaatcgtggtggccggcatgctgctgagaggcgccgtggtgcctgacagcgccctgccattcaattttcaagccgcctacggcctgagcgatcagctggcccaggccatcagcgaccactaccccgtggaagtgatgctgaagtga 98 RSLV133:kesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdv hRNAseWT-qnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiiv SCC-acegspyvpvhfdasvedstlepkssdkthtcppcpapellggssvflfppkpk mthIgG1-dtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrv P238S-vsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsrd P3315-NLG-eltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskl hDNAsetvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqd 105/114ilkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk 99 NLGlnk2vdgasspvnvsspsvqdi 100 VK3LP metpaqllfllllwlpdtt leader 101 hRNaseWTkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdv (mature)qnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiiv UniProtacegspyvpvhfdasvedst P07998) 102 hDNase 1lkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgklldn (mature)lnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndt UniProtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgled P24855vmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk 103 hDNase 1L3mricsfnvrsfgeskqedknamdvivkvikrcdiilvmeikdsnnricpilmek (mature)lnrnsrrgitynyvissrlgrntykeqyaflykeklvsvkrsyhyhdyqdgdad UniProtvfsrepfvvwfqsphtavkdfviiplhttpetsvkeidelvevytdvkhrwkae Q13609nfifmgdfnagcsyvpkkawknirlrtdprfvwligdqedttvkkstncaydrivlrgqeivssvvpksnsvfdfqkayklteeealdvsdhfpvefklqssraftns kksvtlrkktkskrs104 hTREX1 mgpgarrqgrivqgrpemcfcppptplpplriltlgthtptpcsspgsaagtyptmgsqalppgpmqtliffdmeatglpfsqpkvtelcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitglstavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelamlgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartkprpsavtttahlattrntspslgesrgtkdlppvkdpgalsregllaplgllailtlavatlyglslatpge 105 hTREX1 (C-mgpgarrqgrivqgrpemcfcppptplpplriltlgthtptpcsspgsaagtyp terminal 72tmgsqalppgpmqtliffdmeatglpfsqpkvtelcllavhrcalespptsqgp aapptvpppprvvdklslcvapgkacspaaseitglstavlaahgrqcfddnlanl truncated)llaflrrqpqpwclvahngdrydfpllqaelamlgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartk 106 RSLV-124metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycngmmrrrn hVK3LP-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNase(WT)-gsrypncayrtspkerhiivacegspyvpvhfdasvedstlepkssdkthtcpp hIgG1 WTcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhgdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytgkslsls pgk 107 NLGlnkvdgasspvnvsspsvqdi

1. A hybrid nuclease molecule comprising a first nuclease domain and amodified Fc domain, wherein the first nuclease domain is operativelycoupled to the Fc domain and wherein the Fc domain is modified such thatthe molecule has reduced cytotoxicity relative to a hybrid nucleasemolecule having an unmodified Fc domain.